Method and compositions for immunotherapy of inflammatory and immune-dysregulatory diseases

ABSTRACT

Methods and compositions for immunotherapy of inflammatory and immune-dysregulatory diseases, using multispecific antagonists that target at least two different markers are disclosed. The different targets include proinflammatory effectors of the innate immune system and targets specifically associated with an inflammatory or immune-dysregulatory disorder, wherein the targets included in the latter group are not a proinflammatory effector of the immune system. Thus, the multispecific antagonist contains at least one binding specificity related to the diseased cell or condition being treated and at least one specificity to a component of the immune system. The multispecific antagonists are used in the treatment of various diseases that are generated or exacerbated by, or otherwise involve, proinflammatory effectors of the innate immune system.

This application claims the benefit of U.S. Provisional Application No.60/634,076, filed on Dec. 8, 2004.

BACKGROUND OF THE INVENTION

A. Field of the Invention

The invention relates generally to methods and compositions forimmunotherapy of inflammatory and immune-dysregulatory diseases, usingmultispecific antagonists that target at least two different markers.The markers are antigens and/or receptors on lymphocytes, macrophages,monocytes, or dendritic cells (DCs). The invention particularly relatesto methods and compositions for modulating receptors on immune-targetingand immune-processing cells using specific antibodies and antibodyheteroconjugates to bind to the cells and their receptors, to effect atreatment of various diseases that are generated or exacerbated by, orotherwise involve, these cells and their receptors. Such diseases moreparticularly include acute and chronic inflammatory disorders,autoimmune diseases, septicemia and septic shock, neuropathies, graftversus host disease, acute respiratory distress syndrome, granulomatousdiseases, giant cell arteritis, acne, diffuse intravascular coagulation(DIC), transplant rejection, asthma, cachexia, myocardial ischemia, andatherosclerosis. The methods and compositions also are useful intreating pathological angiogenesis and cancer. The methods andcompositions can include a secondary therapeutic that is directed to acancer receptor or cancer-associated antigen. Methods and compositionsare also described for improved diagnosis/detection of the diseases.

B. Description of the Related Art

The immune system comprises both the innate immune system and theadaptive, or acquired immune system. Many host cells participate in theprocesses of innate and adaptive immunity, such as neutrophils, T- andB-lymphocytes, macrophages and monocytes, dendritic cells, and plasmacells. They usually act in concert, affecting one another, particularlyin the regulation of certain factors and cytokines that contribute tothe recognition and processing of innate and external noxients, andthese systems have evolved over the millions of years of the developmentof vertebrate, mammalian, and human organisms.

A major goal of immunotherapy is to exploit or enhance a patient'simmune system against an innate or foreign noxient, such as a malignantcell or an invading microorganism. The immune system has been studiedmore in relation to recognizing and responding to exogenous noxients,such as microbial organisms, than it has in relation to indigenousmalfunctions, such as cancer and certain autoimmune andimmune-dysregulatory diseases, particularly since the latter may haveboth genetic as well as environmental components. The defenses againstmicrobial organisms, such as bacteria, fungi, parasites, and viruses,are innate to the particular organism, with the immune system beingprogrammed to recognize biochemical patterns of these microorganisms andto respond to attack them without requiring prior exposure to themicroorganism. This innate immune system includes, for example,neutrophils, natural killer cells and monocytes/macrophages that caneradicate the invading microorganisms by direct engulfment anddestruction.

The innate immune response is often referred to as a nonspecific onethat controls an invading external noxient until the more specificadaptive immune system can marshal specific antibodies and T cells (cf.Modlin et al., N Engl J Med 1999, 340:1834-1835; Das, Crit Care 2000;4:290-296). The nonspecific immune responses involve the lymphaticsystem and phagocytes. The lymphatic system includes the lymphocytes andmacrophages. Macrophages can engulf, kill and dispose of foreignparticles. Phagocytes include neutrophils and macrophages, which againingest, degrade and dispose of debris, and have receptors for complementand antibody. In summary, the innate immune system provides a line ofdefense again certain antigens because of inherited characteristics.

In contrast, the adaptive, or acquired, immune system, is highly evolvedand very specific in its responses. It is called an adaptive systembecause is occurs during the lifetime of an individual as an adaptationto infection with a pathogen. Adaptive immunity can be artificiallyacquired in response to a vaccine (antigens) or by administeringantibodies, or can be naturally acquired by infection. The acquiredimmunity can be active, if an antibody was produced, or it can bepassive, if exogenous antibody made form another source is injected.

The adaptive immune system produces antibodies specific to a givenantigen. The simplest and most direct way in which antibodies provideprotection is by binding to them and thereby blocking their access tocells that they may infect or destroy. This is known as neutralization.Binding by antibodies, however, is not sufficient to arrest thereplication of bacteria that multiply outside cells. In this case, onerole of antibody is to enable a phagocytic cell to ingest and destroythe bacterium. This is known as opsonization. The third function ofantibodies is to activate a system of plasma proteins, known ascomplement. In many cases, the adaptive immune system confers lifelongprotective immunity to re-infection with the same pathogen, because theadaptive immune system has a ‘memory’ of the antigens presented to it.

Antibody-mediated immunity is called humoral immunity and is regulatedby B cells and the antibodies they produce. Cell-mediated immunity iscontrolled by T cells. Both humoral and cell-mediated immunityparticipate in protecting the host from invading organisms. Thisinterplay can result in an effective killing or control of foreignorganisms. Occasionally, however, the interplay can become erratic. Inthese cases, there is a dysregulation that can cause disease. Sometimesthe disease is life-threatening, such as with septic shock and certainautoimmune disorders.

The B and T lymphocytes are critical components of a specific immuneresponse. B cells are activated by antigen to engender clones ofantigen-specific cells that mediate adaptive immunity. Most clonesdifferentiate to plasma cells that secrete antibody, while a few clonesform memory cells that revert to plasma cells. Upon subsequentre-infection, memory cells produce a higher level of antibody in ashorter period than in the primary response. Antibodies secreted by theplasma cells can play multiple roles in immunity, such as binding andneutralizing a foreign agent, acting as opsonins (IgG) to promotephagocytosis, directly affecting metabolism and growth of someorganisms, engaging in antigen-antibody reactions that activatecomplement, causing phagocytosis and membrane attack complex, and/orengaging in antigen-antibody reactions that activate T cells and otherkiller cells.

T lymphocytes function as both helper cells and suppressor cells. HelperT cells induce antigen-specific B cells and effector T cells toproliferate and differentiate. Suppressor T cells interact with helper Tcells to prevent an immune response or to suppress an ongoing one, or toregulate effector T cells. Cytotoxic T cells destroy antigen by bindingto target cells. In a delayed-type hypersensitivity reaction, the Tcells do not destroy antigen, but attract macrophages, neutrophils andother cells to destroy and dispose of the antigen.

T cells can detect the presence of intracellular pathogens becauseinfected cells display on their surface peptide fragments derived fromthe pathogens' proteins. These foreign peptides are delivered to thecell surface by specialized host-cell glycoproteins, termed MajorHistocompatibility Complex (MHC) molecules. The recognition of antigenas a small peptide fragment bound to a MHC molecule and displayed at thecell surface is one of the most distinctive features of T cells. Thereare two different classes of MHC molecules, know as MHC class I and MHCclass II, that deliver peptides from different cellular compartments tothe surface of the infected cell. Peptides from the cytosol are bound toMHC class I molecules which are expressed on the majority of nucleatedcells and are recognized by CD8+ T cells. MHC class II molecules, incontrast, traffic to lysosomes for sampling endocytosed protein antigenswhich are presented to the CD4+ T cells (Bryant and Ploegh, Curr OpinImmunol 2004; 16:96-102).

CD8+ T cells differentiate into cytotoxic T cells, and kill the cell.CD4+ T cells differentiate into two types of effector T cells. Pathogensthat accumulate in large numbers inside macrophage vesicles tend tostimulate the differentiation of T_(H)1 cells which activate macrophagesand induce B cells to make IgG antibodies that are effective inopsonizing extracellular pathogens for uptake by phagocytes.Extracellular antigens tend to stimulate the production of T_(H)2 cellswhich initiate the humoral immune response by activating naïveantigen-specific B cells to produce IgM antibodies, inter alia.

The innate and adaptive immune systems interact, in that the cells ofthe innate immune system can express various molecules that can interactwith or trigger the adaptive immune system by activating certain cellscapable of producing immune factors, such as by activating T and B cellsof the lymphatic series of leukocytes. The early induced butnon-adaptive responses are important for two main reasons. First, theycan repel a pathogen or, more often, control it until an adaptive immuneresponse can be mounted. Second, these early responses influence theadaptive response in several ways. For example, the innate immuneresponse produces cytokines and other inflammatory mediators that haveprofound effects on subsequent events, including the recruitment of newphagocytic cells to local sites of infection. Another effect of thesemediators is to induce the expression of adhesion molecules on theendothelial cells of the local blood vessels, which bind to the surfaceof circulating monocytes and neutrophils and greatly increase their rateof migration of these cells out of the blood and into the tissues. Theseevents all are included under the term inflammation, which is a featureof the innate immune system that forms part of the protective responseat a localized site to isolate, destroy and remove a foreign material.This is followed by repair. Inflammation is divided into acute andchronic forms.

The immune system communicates via nonspecific tissue resistancefactors. These include the interferons, which are proteins produced inresponse to viruses, endotoxins and certain bacteria. Interferonsinhibit viral replication and activate certain host-defense responses.Infected cells produce interferon that binds the infected cells toother, neighboring cells, causing them to produce antiviral proteins andenzymes that interfere with viral gene transcription and proteinssynthesis. Interferons can also affect normal cell growth and suppresscell-mediated immunity.

Complement is another nonspecific tissue resistance factor, andcomprises plasma proteins and membrane proteins that mediate specificand non-specific defenses. Complement has two pathways, the classicalpathway associated with specific defense, and the alternative pathwaythat is activated in the absence of specific antibody, and is thusnon-specific. In the classical pathway, antigen-antibody complexes arerecognized when C1 interacts with the Fc of the antibody, such as IgMand to some extent, IgG, ultimately causing mast cells to releasechemotactic factors, vascular mediators and a respiratory burst inphagocytes, as one of many mechanisms. The key complement factorsinclude C3a and C5a, which cause mast cells to release chemotacticfactors such as histamine and serotonin that attract phagocytes,antibodies and complement, etc. Other key complement factors are C3b andC5b, which enhance phagocytosis of foreign cells, and C8 and C9, whichinduce lysis of foreign cells (membrane attack complex).

Cancer cells can escape immune surveillance by avoiding complementactivation, especially by the expression of membrane-associatedcomplement regulatory proteins, such as CD55 (decay-acceleratingfactor), CD46 (membrane cofactor protein), and CD59 (protectin), and itis believed that the over-expression of these proteins on cancer cellmembranes protects these cancers from complement activation (Brasoveanuet al., Lab Invest 1996; 74:33-42; Jarvis et al., Int J Cancer 1997;71:1049-1055; Yu et al., Clin Exp Immunol 1999; 115:13-18; Murray etal., Gynecol Oncol 2000; 76:176-182; Donin et al., Clin Exp Immunol2003; 131:254-263). Attempts have been made, unsuccessfully, to increasethe susceptibility to complement-mediated lysis by use of neutralizingantibodies against CD46, CD55 and CD59 (Varsano et al., Clin Exp Immunol1998; 113:173-182 Junnikkala et al., J Immunol 2000; 164:6075-6081;Maenpaa et al., Am J Pathol 1996; 148:1139-1162; Gorter Lab Invest 1996;74:1039-1049. In the latter study, CD46 and CD55 antibodies were, incontrast to CD59 antibodies, ineffective. This suggests that othertargets, or the use of antibodies against multiple complement regulatoryproteins, or against both complement regulatory proteins and othermediators of immunity may be required. This general failure contradictsthe speculation of Fishelson et al. (Mol Immunol 2003: 40:109-123) andthe suggestion from other studies that treatment of cancer patients withantibodies to membrane complement regulatory proteins in combinationwith anticancer complement-fixing antibodies will improve therapeuticefficacy, so there remains a need to elucidate how such strategies maybest be implemented in cancer patients.

Gelderman et al. (Mol Immunol 2003; 40:13-23) reported thatmembrane-bound complement regulatory proteins (mCRP) inhibit complementactivation by an immunotherapeutic mAb in a syngeneic rat colorectalcancer model. While the use of mAb against tumor antigens and mCRPovercame an observed effect of mCRP on tumor cells, there has been nodirect evidence to support this approach. Still other attempts to usebispecific antibodies against CD55 and against a tumor antigen (G250 orEpCAM) have been suggested by Gelderman et al. (Lab Invest 2002;82:483-493; Eur J Immunol 2002; 32:128-135) based on in vitro studiesthat showed a 2-13-fold increase in C3 deposition compared to use of theparental antitumor antibody. However, no results involving enhanced cellkilling were reported. Jurianz et al. (Immunopharmacology 1999;42:209-218) also suggested that combining treatment of a tumor withanti-HER2 antibodies in vitro could be enhanced by prior treatment withantibody-neutralization of membrane-complement-regulatory protein, butagain no in vivo results were provided. Sier et al. (Int J Cancer 2004;109:900-908) recently reported that a bispecific antibody made againstan antigen expressed on renal cell carcinoma (Mab G250) and CD55enhanced killing of renal cancer cells in spheroids when beta-glucan wasadded, suggesting that the presence of CR3-priming beta-glucan wasobligatory.

Neutrophils, another cell involved in innate immune response, alsoingest, degrade and dispose of debris. Neutrophils have receptors forcomplement and antibody. By means of complement-receptor bridges andantibody, the foreign noxients can be captured and presented tophagocytes for engulfment and killing.

Macrophages are white blood cells that are part of the innate systemthat continually search for foreign antigenic substances. As part of theinnate immune response, macrophages engulf, kill and dispose of foreignparticles. However, they also process antigens for presentation to B andT cells, invoking humoral or cell-mediated immune responses.

The dendritic cell is one of the major means by which innate andadaptive immune systems communicate (Reis e Sousa, Curr Opin Immunol2004; 16:21-25). It is believed that these cells shape the adaptiveimmune response by the reactions to microbial molecules or signals.Dendritic cells capture, process and present antigens, thus activatingCD4+ and CD8+ naïve T lymphocytes, leading to the induction of primaryimmune responses, and derive their stimulatory potency from expressionof MHC class I, MHC class II, and accessory molecules, such as CD40,CD54, CD80, CD86, and T-cell activating cytokines (Steinman, J ExpHematol 1996; 24:859-862; Banchereau and Steinman, Nature 1998;392:245-252). These properties have made dendritic cells candidates forimmunotherapy of cancers and infectious diseases (Nestle, Oncogene 2000;19:673-679; Fong and Engleman, Annu Rev Immunol 2000; 18:245-273;Lindquist and Pisa, Med Oncol 2002; 19:197-211), and have been shown toinduce antigen-specific cytotoxic T cells that result in strong immunityto viruses and tumors (Kono et al., Clin Cancer Res 2002; 8:394-40).

Also important for interaction of the innate and adaptive immune systemsis the NK cell, which appears as a lymphocyte but behaves like a part ofthe innate immune system. NK cells have been implicated in the killingof tumor cells as well as essential in the response to viral infections(Lanier, Curr Opin Immunol 2003; 15:308-314; Carayannopoulos andYokoyama, Curr Opin Immunol 2004; 16:26-33). Yet another importantmechanism of the innate immune system is the activation of cytokinemediators that alert other cells of the mammalian host to the presenceof infection, of which a key component is the transcription factor NF-κB(Li and Verma, Nat Rev Immunol 2002; 2:725-734).

As mentioned earlier, the immune system can overreact, resulting inallergies or autoimmune diseases. It can also be suppressed, absent, ordestroyed, resulting in disease and death. When the immune system cannotdistinguish between “self” and “nonself,” it can attach and destroycells and tissues of the body, producing autoimmune diseases, e.g.,juvenile diabetes, multiple sclerosis, myasthenia gravis, systemic lupuserythematosus, rheumatoid arthritis, and immune thrombocytopenicpurpura. Immunodeficiency disease results from the lack or failure ofone or more parts of the immune system, and makes the individualssusceptible to diseases that usually do not affect individuals with anormal immune system. Examples of immunodeficiency disease are severecombined immunodeficiency disease (SCID) and acquired immunodeficiencydisease (AIDS). The latter results from human immunodeficiency virus(HIV) and the former from enzyme or other inherited defects, such asadenosine deaminase deficiency.

A comprehensive description of the immune response and various aspectsof immunity, autoimmune disorders, and immunodeficiency disorders isprovided in Janeway et al., IMMUNOBIOLOGY: THE IMMUNE SYSTEM IN HEALTHAND DISEASE, Current Biology Publications, 1999; the MedicalEncyclopedia of Medline Plus(nlm.nih.gov/medlineplus/encyclopedia.html); and the internet siteslic2.wsu.edu:82:hurlbert/micro101/pages/hap15.html, all of which areincorporated herein in their entirety by reference.

The application of immunotherapy to cancer involves a number ofapproaches to engage or exploit the immune system, such as adoptivetransfer of anti-tumor-reactive T cells and the use of vaccines, as wellas breaking tolerance to tumor self-antigens by inhibiting regulatorycells, and boosting T-cell immunity by use of various cytokines andso-called immune-enhancing molecules (Antonia et al., Curr Opin Immunol2004; 16:130-136). Dendritic-cell vaccines have also been described.Direct and indirect (mediated by host effector cells) actions ofantibodies administered to patients by targeting tumor-cellantigens/receptors have now entered the cancer therapy armamentarium, asexemplified by antibodies against CD20 and CD52 in the therapy oflymphomas and leukemia; anti-epidermal growth factor receptor (EGFR),the anti-HER2/neu variant, in the therapy of diverse solid tumors; andanti-vascular endothelium growth factor (VEGF) for the treatment ofcertain solid tumors. Although active when given alone, most of theseshow enhanced antitumor effects when combined with other treatmentmodalities, such as drugs and radiation. Using these tumor-targetingantibodies to deliver cytotoxic drugs or isotopes is still anothermethod of immunotherapy that has entered the clinic. These and othermethods of cancer immunotherapy have been reviewed in Huber and Wölfel,J Cancer Res Clin Oncol 2004; 130:367-374, incorporated herein in itsentirety by reference. However, at best these approaches show reductionof tumor and improved survival in a proportion of the patients, most ofwhom eventually relapse, thus requiring other therapeutic interventionsand different strategies to control their disease.

Sepsis is a major medical and economic burden to our society, affectingabout 700,000 people annually in the United States, causing over200,000, deaths annually, and costing approximately $16.7 billion peryear (Angus et al., Crit Care Med 2001; 29:1303-1310; Martin et al., NEngl J Med 2003; 348:1546-1554). The definition of sepsis has beendifficult, and historically it was defined as the systemic host responseto an infection. A discussion of the clinical definition of sepsis,encompassing systemic inflammatory response syndrome (SIRS), sepsis perse, severe sepsis, septic shock, and multiple organ dysfunction syndrome(MODS) is contained in Riedmann et al., J Clin Invest 2003; 112:460-467.Since it has been a common belief that sepsis is caused by the host'soverwhelming reaction to the invading microorganisms, and that thepatient is more endangered by this response that than the invadingmicroorganisms, suppression of the immune and inflammatory responses wasan early goal of therapy.

Numerous and diverse methods of immunosuppression or of neutralizingproinflammatory cytokines have proven to be unsuccessful clinically inpatients with sepsis and septic shock anti-inflammatory strategies.(Riedmann, et al., cited above; Van Amersfoort et al. (Clin MicrobiolRev 2003; 16:379-414), such as general immunosuppression, use ofnonsteroidal anti-inflammatory drugs, TNF-α antibody (infliximab) or aTNF-R:Fc fusion protein (etanercept), IL-1 (interleukin-1) receptorantagonist, or high doses of corticosteroids. However, a success in thetreatment of sepsis in adults was the PROWESS study (Human ActivatedProtein C Worldwide Evaluation in Severe Sepsis (Bernard et al., N EnglJ Med 2001; 344:699-709)), showing a lower mortality (24.7%) than in theplacebo group (30.8%). This activated protein C agent probably inhibitsboth thrombosis and inflammation, whereas fibrinolysis is fostered. VanAmersfoort et al. state, in their review (ibid.) that: “Although theblocking or modulation of a number of other targets including complementand coagulation factors, neutrophil adherence, and NO release, arepromising in animals, it remains to be determined whether thesetherapeutic approaches will be effective in humans.” This is furtheremphasized in a review by Abraham, “Why immunomodulatory therapies havenot worked in sepsis” (Intensive Care Med 1999; 25:556-566).

The immune system in sepsis is believed to have an early intenseproinflammatory response after infection or trauma, leading to organdamage, but it is also believed that the innate immune system oftenfails to effectively kill invading microorganisms (Riedmann and Ward,Expert Opin Biol Ther 2003; 3:339-350). There have been some studies ofmacrophage migration inhibitory factor (MIF) in connection with sepsisthat have shown some promise. For example, blockage of MIF or targeteddisruption of the MIF gene significantly improved survival in a model ofseptic shock in mice (Calandra et al., Nature Med 2000; 6:164-170), andseveral lines of evidence have pointed to MIF as a potential target fortherapeutic intervention in septic patients (Riedmann et al., citedabove). Bucala et al. (U.S. Pat. No. 6,645,493 B1) have claimed that ananti-MIF antibody can be effective therapeutically for treating acondition or disease caused by cytokine-mediated toxicity, includingdifferent forms of sepsis, inflammatory diseases, acute respiratorydisease syndrome, granulomatous diseases, chronic infections, transplantrejection, cachexia, asthma, viral infections, parasitic infections,malaria, and bacterial infections, which is incorporated herein in itsentirety, including references. The use of anti-LPS (lipopolysaccharide)antibodies alone similarly has had mixed results in the treatment ofpatients with septic shock (Astiz and Rackow, Lancet 1998;351:1501-1505; Van Amersfoort et al., Clin Microbiol Rev 2003;16:379-414.

While both LPS and MIF have been pursued as targets in the treatment ofsepsis and septic shock, approaches which target LPS or MIF alone by anantibody have not been sufficient to control the diverse manifestationsof sepsis, especially in advanced and severe forms. Similarly, use ofcytokines, such as IL-1, IL-6 (interleukin-6), IL-8 (interleukin-8),etc., as targets for antibodies for the treatment of sepsis and othercytokine-mediated toxic reactions, has not proven to be sufficient for ameaningful control of this disease. Therefore, in addition to the needto discover additional targets of the cytokine cascade involved in theendogenous response in sepsis, it has now been discovered that bi- andmulti-functional antibodies targeting at least one cytokine or causativeagent, such as MIF or lipopolysaccharide (LPS), is advantageous,especially when combined with the binding to a host cell (or itsreceptor) engaged in the inflammatory or immune response, such as Tcells, macrophages or dendritic cells. Antibodies against an MHC classII invariant chain target, such as CD74, have been proposed by Bucala etal. (US 2003/0013122 A1), for treating MIF-regulated diseases, andHansen et al. (US 2004/0115193 A1) proposed at least one CD74 antibodyfor treating an immune dysregulation disease, an autoimmune disease,organ graft rejection, and graft versus host disease. Hansen et al.describe the use of fusion proteins of anti-CD74 with other antibodiesreacting with antigens/receptors on host cells such as lymphocytes andmacrophages for the treatment of such diseases. However, combinationswith targets other than CD74 are not suggested, and the disclosurefocuses on a different method of immunotherapy. Similar targets are alsouseful to treat atherosclerotic plaques (Burger-Kentischer et al.,Circulation 2002; 105:1561-1566).

In the treatment of infectious, autoimmune, organ transplantation,inflammatory, and graft versus host (and other immunoregulatory)diseases, diverse and relatively non-specific cytotoxic agents are usedto either kill or eliminate the noxient or microorganism, or to depressthe host's immune response to a foreign graft or immunogen, or thehost's production of antibodies against “self,” etc. However, theseusually affect the lymphoid and other parts of the hematopoietic system,giving rise to toxic effects to the bone marrow (hematopoietic) andother normal host cells.

Therefore, a need exists for improved, more selective therapy of cancerand diverse immune diseases, including sepsis and septic shock,inflammation, atherosclerosis, cachexia, graft versus host, and otherimmune dysregulatory disorders. The present invention is directed toovercoming or at least reducing the effects of one or more of theproblems set forth above.

SUMMARY OF THE INVENTION

The present invention provides new and well-tolerated methods which usemultispecific antagonists in the therapy of various inflammatory andimmune-dysregulatory diseases, infectious diseases, pathologicangiogenesis and cancer. The multispecific antagonists are moreeffective than agents which react specifically with only one targetassociated with these conditions. The present invention provides amultispecific antagonist that reacts specifically with at least twodifferent targets. The targets are selected from the group consisting of(A) proinflammatory effectors of the innate immune system, (B)coagulation factors, (C) complement factors and complement regulatoryproteins, and (D) targets specifically associated with an inflammatoryor immune-dysregulatory disorder or with a pathologic angiogenesis orcancer, wherein the latter target is not (A), (B), or (C). At least oneof the targets is (A), (B) or (C). When the multispecific antagonistcomprises a single multispecific antibody, then CD74 is excluded as atarget of the antagonist. Furthermore, when the multispecific antagonistcomprises a combination of separate antibodies, combinations areexcluded where one of the antibodies targets a B-cell antigen and theother antibody targets a T-cell, plasma cell, macrophage or inflammatorycytokine. Combinations of separate antibodies are also excluded whereone of the antibodies targets CD20 and the other antibody targets C3b orCD40.

The term “reacts specifically” encompasses not only the binding of anantibody or antibody fragment to an antigen, but also to the binding ofa receptor to its cognate ligand. For example, a receptor for aproinflammatory effector of the innate immune system “reactsspecifically” with its proinflammatory effector cognate ligand and thusfalls within the scope of the present invention. In some embodiments,the multispecific antagonist is a combination of two separateantibodies. In other embodiments, it is a multispecific antibody,particularly a fusion protein.

In one embodiment, when the multispecific antagonist comprises acombination of separate antibodies, combinations are excluded where oneof the antibodies targets CD19, CD20, CD21, CD22, CD23 or CD80 and theother antibody targets a complement factor. More particularly, when themultispecific antagonist comprises a combination of separate antibodies,combinations are excluded where one of the antibodies targets CD19,CD20, CD21, CD22, CD23 or CD80 and the other antibody targets C3b orCD40.

The proinflammatory effector of the innate immune system may be aproinflammatory effector cytokine, a proinflammatory effector chemokineor a proinflammatory effector receptor. Suitable proinflammatoryeffector cytokine include MIF, HMGB-1 (high mobility group box protein1), TNF-α, IL-1, IL-4 (interleukin-4), IL-5 (interleukin-5), IL-6, IL-8,IL-12 (interleukin-12), IL-15 (interleukin-15), IL-17 (interleukin-17),and IL-18 (interleukin-18). Examples of proinflammatory effectorchemokines include CCL19, CCL21, IL-8, MCP-1, RANTES, MIP-1A, MIP-1B,ENA-78, MCP-1, IP-10, GROB, and Eotaxin. Proinflammatory effectorreceptors include IL-4R (interleukin-4 receptor), IL-6R (interleukin-6receptor), IL-13R (interleukin-13 receptor), IL-15R (interleukin-15receptor), IL-17R (interleukin-17 receptor) and IL-18R (interleukin-18receptor).

The multispecific antagonist also may react specifically with at leastone coagulation factor, particularly tissue factor (TF) or thrombin. Inother embodiments, the multispecific antagonist reacts specifically withat least one complement factor or complement regulatory protein. Inpreferred embodiments, the complement factor is selected from the groupconsisting of C3, C5, C3a, C3b, and C5a. In these embodiments, targetcombinations preferably do not include those in which the other antibodytargets CD19, CD20, CD21, CD22, CD23 or CD80 when the antagonist is acombination of separate antibodies. When the antagonist reactsspecifically with a complement regulatory protein, the complementregulatory protein preferably is selected from the group consisting ofCD46, CD55, CD59 and mCRP.

In one embodiment, the multispecific antagonist comprises two or moreantibodies which differ in specificity, each of which reactsspecifically with a different proinflammatory effector of the innateimmune system. Alternatively, the multispecific antagonist comprises twoor more antibodies that differ in specificity, each of which reactsspecifically with a different coagulation factor. In another embodiment,the multispecific antagonist comprises two or more antibodies thatdiffer in specificity, each of which reacts specifically with adifferent complement factor or complement regulatory protein. In yetother embodiments, the two or more antibodies react specifically with atleast one proinflammatory effector of the innate immune system and withat least one coagulation factor, or with at least one proinflammatoryeffector of the innate immune system and with at least one complementfactor or complement regulatory protein, or with at least one complementfactor or complement regulatory protein and with at least onecoagulation factor, respectively. Alternatively, the multispecificantagonist may react specifically with more than one proinflammatoryeffector of the innate immune system, or with more than one coagulationfactor, or with more than one complement factor or complement regulatoryprotein.

The two or more antibodies may react specifically with more than oneepitope of the same proinflammatory effector of the innate immune systemor more than one epitope of the same coagulation factor or more than oneepitope of the same complement factor or complement regulatory protein.In any of these embodiments, the multispecific antagonist additionallymay react with a target specifically associated with an inflammatory orimmune-dysregulatory disorder or with a pathologic angiogenesis orcancer, which target is not an (A), (B) or (C) target as defined above.In other embodiments, the multispecific antagonist reacts with a targetspecifically associated with an inflammatory or immune-dysregulatorydisorder or with a pathologic angiogenesis or cancer, and with one ormore (A), (B) or (C) targets as defined above. An example of a usefultarget for pathologic angiogenesis is Flt-1.

In any of the embodiments of the invention, the multispecific antagonistmay be a multispecific antibody in which different arms of the antibodyreact with the different targets, subject to provisos elucidated herein.Such multispecific constructs can also have multivalency in any or allbinding arms against the same antigen epitope in order to enhancebinding to the antigen target, as described in Rossi (Patent ApplicationWO 04094613A2).

The multispecific antagonist alternatively may comprise at least onesoluble receptor, or at least an extracellular domain of at least oneproinflammatory effector receptor. In one embodiment, the antagonistcomprises at least one soluble receptor or at least an extracellulardomain of a proinflammatory effector receptor fused to at least oneantibody.

The multispecific antagonist may comprise at least one molecule reactivewith a proinflammatory effector receptor. This molecule preferably is anatural antagonist for the proinflammatory effector receptor, or afragment or mutant of the antagonist that interacts specifically withthe receptor. In one embodiment, the natural antagonist is the naturalIL-1 receptor antagonist, or a fragment or mutant of this antagonist.

The multispecific antagonist additionally may target dendritic cells,granulocytes, monocytes, macrophages, NK-cells, platelets, orendothelial cells. In some embodiments, the multispecific antagonistspecifically reacts with at least one antigen or receptor of theadaptive immune system. In other embodiments, the multispecificantagonist specifically reacts with a cancer cell receptor orcancer-associated antigen, such as B-cell lineage antigens (CD19, CD20,CD21, CD22, CD23, etc.), VEGFR, EGFR, carcinoembryonic antigen (CEA),placental growth factor (PLGF), tenascin, HER-2/neu, EGP-1, EGP-2, CD25,CD30, CD33, CD38, CD40, CD45, CD52, CD74, CD80, CD138, NCA66, MUC1,MUC2, MUC3, MUC4, MUC16, IL-6, α-fetoprotein (AFP), A33, CA125,colon-specific antigen-p (CSAp), folate receptor, HLA-DR, humanchorionic gonadotropin (HCG), Ia, EL-2, insulin-like growth factor(ILGF) and ILGF receptor, KS-1, Le(y), MAGE, necrosis antigens, PAM-4,prostatic acid phosphatase (PAP), Pr1, prostate specific antigen (PSA),PSMA, S100, T101, TAC, TAG72, TRAIL receptors, and carbonic anhydraseIX. Flt-3, which targets proliferating myeloid bone marrow cells, alsois a useful in identifying and treating certain cancers. Alternatively,the multispecific antagonist may react specifically with a target suchas C5a, Factor H, FHL-1, LPS, IFNγ or B7, or with a target such as CD2,CD4, CD14, CD18, CD11a, CD19, CD20, CD22, CD23, CD25, CD29, CD38, CD40L,CD52, CD64, CD83, CD147 or CD154.

The multispecific antagonist may comprise a single active component, orit may comprise multiple active components. The embodiment comprising asingle active component does not encompass mixtures of antibodies, whichwould by definition comprise two or more active components.Multispecific antagonists comprising more than one active component alsomay include secondary therapeutics that affect a component of the innateimmune system, a component of the adaptive immune system, coagulation,infectious agents or cancer cells.

The multispecific antagonist may react specifically with targets ormarkers associated with specific diseases and conditions, such asinfectious diseases, acute respiratory distress syndrome, septicemia orseptic shock, graft versus host disease or transplant rejection,atherosclerosis, asthma, acne, giant cell arteritis, a granulomatousdisease, a neuropathy, cachexia, a coagulopathy such as diffuseintravascular coagulation (DIC), or myocardial ischemia.

The multispecific antagonists are useful in treating conditions such asinflammatory or immune-dysregulatory disorders, pathologic angiogenesisor cancer, and infectious disease. Treatment comprises administering atherapeutically effective amount of the multi specific antagonist to apatient that has been diagnosed with one of the conditions. In oneembodiment, the inflammatory or immune-dysregulatory disorder is not anautoimmune disease. The multispecific antagonist can be used to treatsepticemia or septic shock, infectious disease (bacterial, viral,fungal, or parasitic), neuropathy, graft versus host disease ortransplant rejection, acute respiratory distress syndrome, agranulomatous disease, asthma, atherosclerosis, acne, giant cellarteritis, coagulopathies such as diffuse intravascular coagulation(DIC), or cachexia. In other embodiments, the condition is an autoimmunedisease, especially a Class III autoimmune diseases.

The multispecific antagonist also can be used to treat a pathologicangiogenesis or cancer. The cancer may be hematopoietic cancer, such asleukemia, lymphoma, or myeloma, etc. Alternatively, the cancer may be asolid tumor, such as a carcinoma, melanoma, sarcoma, glioma, etc.

The multispecific antagonist may be an immunoconjugate that comprises atherapeutic agent, such as a radionuclide, an immunomodulator, ahormone, a hormone antagonist, an enzyme, an enzyme inhibitor,oligonucleotide, a photoactive therapeutic agent, a cytotoxic agent, anantibody, an angiogenesis inhibitor, and a combination thereof. When thetherapeutic agent is an oligonucleotide it may be an antisenseoligonucleotide.

In other embodiments, the therapeutic agent is a cytotoxic agent, forexample, a drug or a toxin. The drug may possess the pharmaceuticalproperty selected from the group consisting of antimitotic, alkylating,antimetabolite, antiangiogenic, apoptotic, alkaloid, proteasomeinhibitor, and antibiotic agents and combinations thereof. In certainembodiments, the drug is selected from the group consisting of nitrogenmustards, gemcitabine, ethylenimine derivatives, alkyl sulfonates,nitrosoureas, triazenes, folic acid analogs, anthracyclines, SN-38,taxanes, COX-2 inhibitors, pyrimidine analogs (e.g., 5-fluorouracil),purine analogs, antibiotics, enzymes, enzyme inhibitors, proteasomeinhibitors, epipodophyllotoxins, platinum coordination complexes, vincaalkaloids, substituted ureas, methyl hydrazine derivatives,adrenocortical suppressants, hormone antagonists, endostatin, taxols,camptothecins, doxorubicins and their analogs, antimetabolites,alkylating agents, antimitotics, antiangiogenic, apoptotoic agents,methotrexate, CPT-11, and a combination thereof. The toxin may bederived from a source selected from the group comprising an animal, aplant, and a microbial source, and is preferably selected from the groupconsisting of ricin, abrin, alpha toxin, saporin, ribonuclease (RNase),DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein,gelonin, diphtherin toxin, Pseudomonas exotoxin, and Pseudomonasendotoxin.

In other embodiments the therapeutic agent is an immunomodulator, suchas a cytokine, a stem cell growth factor, a lymphotoxin, a hematopoieticfactor, a colony stimulating factor (CSF), an interferon (IFN), a stemcell growth factor, erythropoietin, thrombopoietin and a combinationthereof. The lymphotoxin may be tumor necrosis factor (TNF), thehematopoietic factor may be an interleukin (IL), the colony stimulatingfactor may be granulocyte-colony stimulating factor (G-CSF) orgranulocyte macrophage-colony stimulating factor (GM-CSF)), theinterferon may be interferon-α, β or γ, and the stem cell growth factormay be S1 factor. Preferably the immunomodulator comprises IL-1, IL-2,IL-3, IL-6, IL-10, IL-12, IL-17, IL-18, IL-21, interferon-γ, TNF-α, or acombination thereof.

Alternatively, the therapeutic agent is a radionuclide. Preferably theradionuclide has an energy between 60 and 700 keV, an preferably isselected from the group consisting of ³²P, ³³P, ⁴⁷SC ¹²⁵I, ¹³¹I, ⁸⁶Y,⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ¹¹¹In, ¹¹¹Ag, ¹⁴²Pr, ¹⁵³Sm,¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁹⁸Au, ²¹¹At, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²²³Ra and²²⁵Ac, and combinations thereof.

In other embodiments, the therapeutic agent is a photoactive therapeuticagent, such as a chromogen or and dye. The therapeutic agent also may bean enzyme. The enzyme preferably is selected from the group comprisingmalate dehydrogenase, staphylococcal nuclease, delta-V-steroidisomerase, yeast alcohol dehydrogenase, α-glycerophosphatedehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase and acetylcholinesterase.

The multispecific antagonist may comprise a diagnostic/detection agent.The diagnostic/detection agent may be selected from the group consistingof a diagnostic radionuclide, a contrast agent, a fluorescent label, anda photoactive agent.

The invention also provides a method of diagnosing or detecting acondition selected from an inflammatory or immune-dysregulatorydisorder, a pathologic angiogenesis or cancer, and an infectiousdisease, comprising administering a diagnostically effective amount of amultispecific antagonist according to the invention to a patient that issuspected of having such a condition; permitting the multispecificantagonist to accrete at target sites; waiting for circulatingmultispecific antibody to clear from the bloodstream or using a clearingagent; and locating the sites of accretion of said labeled multispecificantagonist by detecting elevated levels of said labeled multispecificantagonist at such sites with a detection means.

Another method of diagnosing or detecting a condition selected from aninflammatory or immune-dysregulatory disorders, a pathologicangiogenesis or cancer, and an infectious disease, comprisesadministering a diagnostically effective amount of a multi specificantagonist according to the invention that includes a hapten bindingsite, to a patient that is suspected of having such a condition;permitting the multi specific antagonist to accrete at target sites;waiting for circulating multispecific antibody to clear from thebloodstream; administering to said subject a hapten labeled with adiagnostic/detection agent; allowing the labeled hapten to bind to thehapten binding site of said multispecific antagonist; and locating thesites of accretion of said multi specific antagonist by detectingelevated levels of said multispecific antagonist bound to said labeledhapten at such sites with a detection means.

The present invention also provides a method of treating a conditionselected from an inflammatory or immune-dysregulatory disorders, apathologic angiogenesis or cancer, and an infectious disease, comprisingadministering a therapeutically effective amount of a multi specificantagonist according to the invention, that includes a hapten bindingsite, to a patient that is suspected of having such a condition;permitting the multi specific antagonist to accrete at target sites;waiting for circulating multispecific antibody to clear from thebloodstream; administering to said subject a hapten that comprises atherapeutic agent; and allowing the hapten with the therapeutic agent tobind to the hapten binding site of said multi specific antagonist.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS Definitions

In the description that follows, and in documents incorporated byreference, a number of terms are used extensively. The followingdefinitions are provided to facilitate understanding of the invention.

A structural gene is a DNA sequence that is transcribed into messengerRNA (mRNA) which is then translated into a sequence of amino acidscharacteristic of a specific polypeptide.

A promoter is a DNA sequence that directs the transcription of astructural gene. Typically, a promoter is located in the 5′ region of agene, proximal to the transcriptional start site of a structural gene.If a promoter is an inducible promoter, then the rate of transcriptionincreases in response to an inducing agent. In contrast, the rate oftranscription is not regulated by an inducing agent when the promoter isa constitutive promoter.

An isolated DNA molecule is a fragment of DNA that is not integrated inthe genomic DNA of an organism. For example, a cloned antibody gene is aDNA fragment that has been separated from the genomic DNA of a mammaliancell. Another example of an isolated DNA molecule is a chemicallysynthesized DNA molecule that is not integrated in the genomic DNA of anorganism.

An enhancer is a DNA regulatory element that can increase the efficiencyof transcription, regardless of the distance or orientation of theenhancer relative to the start site of transcription.

Complementary DNA (cDNA) is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand.

The term expression refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

A cloning vector is a DNA molecule, such as a plasmid, cosmid, orbacteriophage that has the capability of replicating autonomously in ahost cell. Cloning vectors typically contain one or a small number ofrestriction endonuclease recognition sites at which foreign DNAsequences can be inserted in a determinable fashion without loss of anessential biological function of the vector, as well as a marker genethat is suitable for use in the identification and selection of cellstransformed with the cloning vector. Marker genes typically includegenes that provide tetracycline resistance or ampicillin resistance.

An expression vector is a DNA molecule comprising a gene that isexpressed in a host cell. Typically, gene expression is placed under thecontrol of certain regulatory elements, including constitutive orinducible promoters, tissue-specific regulatory elements, and enhancers.Such a gene is said to be “operably linked to” the regulatory elements.

A recombinant host may be any prokaryotic or eukaryotic cell thatcontains either a cloning vector or expression vector. This term alsoincludes those prokaryotic or eukaryotic cells that have beengenetically engineered to contain the cloned gene(s) in the chromosomeor genome of the host cell.

As used herein, antibody encompasses naked antibodies and conjugatedantibodies and antibody fragments, which may be monospecific ormultispecific. It includes recombinant antibodies, such as chimericantibodies, humanized antibodies and fusion proteins.

A chimeric antibody is a recombinant protein that contains the variabledomains and complementary determining regions derived from a rodentantibody, while the remainder of the antibody molecule is derived from ahuman antibody.

Humanized antibodies are recombinant proteins in which murinecomplementarity determining regions of a monoclonal antibody have beentransferred from heavy and light variable chains of the murineimmunoglobulin into a human variable domain. A humanized murine antibody(CDR-grafted) has the murine CDRs grafted into the FRs of a human IgG.The CDR-grafted human variable chains are fused to the constant regionsof a human antibody to obtain an intact humanized IgG.

Human antibodies are antibodies that either are isolated from humans andthen grown out in culture or are made using animals whose immune systemshave been altered so that they respond to antigen stimulation byproducing human antibodies.

An antibody fragment is a portion of an intact antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, sFv and the like. Regardless of structure, anantibody fragment binds with the same antigen that is recognized by thefull-length antibody. For example, an anti-CD20 monoclonal antibodyfragment binds with an epitope of CD20. The term “antibody fragment”also includes any synthetic or genetically engineered protein that actslike an antibody by binding to a specific antigen to form a complex. Forexample, antibody fragments include isolated fragments consisting of thevariable regions, such as the “Fv” fragments consisting of the variableregions of the heavy and light chains, recombinant single chainpolypeptide molecules in which light and heavy variable regions areconnected by a peptide linker (“scFv proteins”), and minimal recognitionunits consisting of the amino acid residues that mimic the hypervariableregion.

Antibody fragments produced by limited proteolysis of wildtypeantibodies are called proteolytic antibody fragments. These include, butare not limited to, the following:

F(ab′)₂ fragments are released from an antibody by limited exposure ofthe antibody to a proteolytic enzyme, e.g., pepsin or ficin. A F(ab′)₂fragment comprises two “arms,” each of which comprises a variable regionthat is directed to and specifically binds a common antigen. The twoFab′ molecules are joined by interchain disulfide bonds in the hingeregions of the heavy chains; the Fab′ molecules may be directed towardthe same (bivalent) or different (bispecific) epitopes.

Fab′ fragments contain a single anti-binding domain comprising a Fab andan additional portion of the heavy chain through the hinge region.

Fab′-SH fragments are typically produced from F(ab′)₂ fragments, whichare held together by disulfide bond(s) between the H chains in anF(ab′)₂ fragment. Treatment with a mild reducing agent such as, by wayof non-limiting example, beta-mercaptoethylamine, breaks the disulfidebond(s), and two Fab′ fragments are released from one F(ab′)₂ fragment.Fab′-SH fragments are monovalent and monospecific.

Fab fragments (i.e., an antibody fragment that contains theantigen-binding domain and comprises a light chain and part of a heavychain bridged by a disulfide bond) are produced by papain digestion ofintact antibodies. A convenient method is to use papain immobilized on aresin so that the enzyme can be easily removed and the digestionterminated. Fab fragments do not have the disulfide bond(s) between theH chains present in an F(ab′)₂ fragment.

Single-chain antibodies are one type of antibody fragment. The termsingle chain antibody is often abbreviated as “scFv” or “sFv.” Theseantibody fragments are produced using molecular genetics and recombinantDNA technology. A single-chain antibody consists of a polypeptide chainthat comprises both a V_(H) and a V_(L) domains which interact to forman antigen-binding site. The VH and VL domains are usually linked by apeptide of 10 to 25 amino acid residues. The term “single-chainantibody” further includes, but is not limited to, a disulfide-linked Fv(dsFv) in which two single-chain antibodies (each of which may bedirected to a different epitope) are linked together by a disulfidebond; a bispecific sFv in which two discrete scFvs of differentspecificity is connected with a peptide linker; a diabody (a dimerizedsFv formed when the V_(H) domain of a first sFv assembles with the V_(L)domain of a second sFv and the V_(L) domain of the first sFv assembleswith the V_(H) domain of the second sFv; the two antigen-binding regionsof the diabody may be directed towards the same or different epitopes);and a triabody (a trimerized sFv, formed in a manner similar to adiabody, but in which three antigen-binding domains are created in asingle complex; the three antigen binding domains may be directedtowards the same or different epitopes). Thus, when making a scFv,diabody, or triabody, the constant regions are not used, and thehumanized variable regions are joined with a linker.

Complementary determining region peptides or CDR peptides are anotherform of an antibody fragment. A CDR peptide (also known as “minimalrecognition unit”) is a peptide corresponding to a singlecomplementarity-determining region (CDR), and can be prepared byconstructing genes encoding the CDR of an antibody of interest. Suchgenes are prepared, for example, by using the polymerase chain reactionto synthesize the variable region from RNA of antibody-producing cells.See, for example, Larrick et al., Methods: A Companion to Methods inEnzymology 2:106, 1991.

In cysteine-modified antibodies, a cysteine amino acid is inserted orsubstituted on the surface of antibody by genetic manipulation and usedto conjugate the antibody to another molecule via, e.g., a disulfidebridge. Cysteine substitutions or insertions for antibodies have beendescribed (see U.S. Pat. No. 5,219,996). Methods for introducing Cysresidues into the constant region of the IgG antibodies for use insite-specific conjugation of antibodies are described by Stimmel et al.(J. Biol. Chem 275:330445-30450, 2000).

As used herein, a therapeutic agent is a molecule or atom, which isconjugated to an antibody moiety to produce a conjugate which is usefulfor therapy. These can be active when given unconjugated to an antibody,such as with ¹³¹I in thyroid neoplasms, and various cytotoxic drugs incancer, autoimmune diseases, graft versus host disease, and in theimmunosuppression induced for organ transplantation, etc. Examples oftherapeutic agents include a therapeutic radionuclide, a boron compound,an immunomodulator, a hormone, a hormone antagonist, an enzyme,oligonucleotides, an enzyme inhibitor, a photoactive therapeutic agent,a cytotoxic agent, and an angiogenesis inhibitor, and a combinationthereof, and these are described in US Published Application no. 20040057902. Preferred therapeutic radioisotopes include beta, alpha, andAuger emitters, with a keV range of 80-500 keV. Exemplary therapeuticradioisotopes include ²²⁵Ac, ¹⁷⁷Lu, ¹⁹⁸Au, ³²P, ¹²⁵I, ¹³¹I ⁹⁰Y, ¹⁸⁶Re,¹⁸⁸Re, ⁶⁷Cu, ⁶⁷Ga, ¹¹¹In, and ²¹¹At.

A diagnostic/detection agent is a molecule or atom which is administeredconjugated to a multispecific antagonist according to the invention,i.e., antibody or antibody fragment, or subfragment, and is useful indiagnosing a disease by locating the cells containing the antigen.Useful diagnostic/detection agents include, but are not limited to,radioisotopes, dyes (such as with the biotin-streptavidin complex),contrast agents, fluorescent compounds or molecules and enhancing agents(e.g., paramagnetic ions) for magnetic resonance imaging (MRI), as wellas for ultrasound and computed tomography.

A naked antibody is an antibody which is not conjugated with atherapeutic agent. Naked antibodies include both polyclonal andmonoclonal antibodies, as well as certain recombinant antibodies, suchas chimeric and humanized antibodies.

A conjugated antibody is an antibody or antibody fragment that isconjugated to a diagnostic or therapeutic agent.

A multispecific antibody is an antibody which can bind simultaneously toat least two targets which are of different structure, e.g., twodifferent antigens, two different epitopes on the same antigen, or ahapten and/or an antigen or epitope. Two or more of the binding arms maybe directed to the same or different epitopes of the same antigen; thusconstituting multivalency in addition to multispecificity.

A bispecific antibody is an antibody or antibody fragment constructwhich can bind simultaneously to two targets which are of differentstructure.

A fusion protein is a recombinantly produced antigen-binding molecule inwhich two or more different single-chain antibody or antibody fragmentsegments with the same or different specificities are linked. A varietyof bispecific fusion proteins can be produced using molecularengineering. In one form, the bispecific fusion protein is monovalent,consisting of, for example, a scFv with a single binding site for oneantigen and a Fab fragment with a single binding site for a secondantigen. In another form, the bispecific fusion protein is divalent,consisting of, for example, an IgG with two binding sites for oneantigen and two scFv with two binding sites for a second antigen.

An infectious disease is one that is caused by a microbe or parasite.

A microbe is a virus, bacteria, rickettsia, mycoplasma, fungi or likemicroorganisms.

A parasite is an infectious, generally microscopic or very small,multicellular invertebrate, protozoan, or an ovum or juvenile formthereof, which is susceptible to antibody-induced clearance or lytic orphagocytic destruction.

Multispecific Antagonists

The present invention provides multispecific antagonists that reactspecifically with at least two different targets. The different targetsinclude proinflammatory effectors of the innate immune system,coagulation factors, complement factors and complement regulatoryproteins, and targets specifically associated with an inflammatory orimmune-dysregulatory disorder, with an infectious pathogen, or with apathologic angiogenesis or cancer, wherein this latter class of targetis not a proinflammatory effector of the immune system or a coagulationfactor. When the multispecific antagonist reacts specifically with atarget associated with an inflammatory or immune-dysregulatory disorder,with a pathologic angiogenesis or cancer, or with an infectious disease,it also binds specifically with at least one proinflammatory effector ofthe immune system, at least one coagulation factor, or at least onecomplement factor or complement regulatory protein. Thus, themultispecific antagonist contains at least one binding specificityrelated to the diseased cell, pathologic angiogenesis or cancer, orinfectious disease, and at least one specificity to a component of theimmune system, such as a receptor or antigen of B cells, T cells,neutrophils, monocytes and macrophages, and dendritic cells, ormodulators of coagulation, such as thrombin or tissue factor, orproinflammatory cytokines, such as IL-1, IL-6, IL-10, HMGB-1, and MIF.

When the multispecific antagonist comprises a single multispecificantibody, then CD74 is excluded as a target of said antagonist.Furthermore, when the multi specific antagonist comprises a combinationof separate antibodies, combinations are excluded where one of thecomponents targets a B-cell antigen, and the other component targets aT-cell, plasma cell, macrophage or inflammatory cytokine.

The present invention is directed to compositions that containmultifunctional proteins and antibodies, and fragments thereof, as wellas to compositions that contain a combination of multiple separateproteins or antibodies, or fragments thereof. Thus, in one embodiment,the multispecific antagonist is an antibody fusion protein or aheteroconjugate. In an alternative embodiment, the multispecificantagonist is an antibody mixture that contains at least two separateantibodies that bind to the different targets. In this embodiment, twoor more antibodies or antibody conjugates are given simultaneously orsequentially. The multispecific antagonist can be naked, but can also beconjugated to a diagnostic imaging agent (e.g., isotope, radiologicalcontrast agent,) or to a therapeutic agent, including a radionuclide, aboron compound, an immunomodulator, a hormone, a hormone antagonist, anenzyme, oligonucleotides, an enzyme inhibitor, a photoactive therapeuticagent, a cytotoxic agent, an angiogenesis inhibitor, and a combinationthereof. The binding of the multispecific antagonist to a target candown-regulate or otherwise affect an immune cell function, but themultispecific antagonist also may bind to other targets that do notdirectly affect immune cell function. For example, an anti-granulocyteantibody, such as against CD66 or CEACAM6 (e.g., NCA90 or NCA95), can beused to target granulocytes in infected tissues, and can also be used totarget cancers that express CEACAM6.

In one embodiment, the therapeutic agent is an oligonucleotide. Forexample, the oligonucleotide can be an antisense oligonucleotide, or adouble stranded interfering RNA (RNAi) molecule. The oligonucleotide canbe against an oncogene like bcl-2 or p53. An antisense moleculeinhibiting bcl-2 expression is described in U.S. Pat. No. 5,734,033. Itmay be conjugated to, or form the therapeutic agent portion of amultispecific antagonist of the present invention. Alternatively, theoligonucleotide may be administered concurrently or sequentially withthe multispecific antagonist of the present invention.

In another embodiment, the therapeutic agent is a boron addend, andtreatment entails irradiation with thermal or epithermal neutrons afterlocalization of the therapeutic agent. The therapeutic agent also may bea photoactive therapeutic agent, particularly one that is a chromogen ora dye.

In a preferred embodiment, the therapeutic agent is a cytotoxic agent,such as a drug or toxin. Also preferred, the drug is selected from thegroup consisting of nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas, gemcitabine, triazenes, folic acid analogs,anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purineanalogs, antibiotics, enzymes, enzyme inhibitors, epipodophyllotoxins,platinum coordination complexes, vinca alkaloids, substituted ureas,methyl hydrazine derivatives, adrenocortical suppressants, hormoneantagonists, endostatin, taxols, SN-38, camptothecins, doxorubicins andtheir analogs, antimetabolites, alkylating agents, antimitotics,antiangiogenic, apoptotoic agents, methotrexate, CPT-11, and acombination thereof.

In another preferred embodiment, the therapeutic agent is a toxinderived from a source selected from the group comprising an animal, aplant, and a microbial source. Preferred toxins include ricin, abrin,alpha toxin, saporin, ribonuclease (RNase), DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin,Pseudomonas exotoxin, and Pseudomonas endotoxins.

The therapeutic agent may be an immunomodulator, such as a cytokine, astem cell growth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), a stem cell growthfactor, erythropoietin, thrombopoietin and a combination thereof saidlymphotoxin is tumor necrosis factor (TNF). The hematopoietic factor maybe an interleukin (IL), the colony stimulating factor may be agranulocyte-colony stimulating factor (G-CSF) or granulocytemacrophage-colony stimulating factor (GM-CSF)), the interferon may beinterferons-α, β or γ, and the stem cell growth factor may be S1 factor.Alternatively, the immunomodulator may comprise IL-1, IL-2, IL-3, IL-6,IL-10, IL-12, IL-17, IL-18, IL-21, interferon-γ, TNF-α, or a combinationthereof.

Preferred therapeutic radionuclides include beta, alpha, and Augeremitters, with a keV range of 80-500 keV. Exemplary therapeuticradioisotopes include ³²P, ³³P, ⁴⁷Sc, ¹²⁵I, ¹³¹I, ⁸⁶Y, ⁹⁰Y, ¹⁸⁶Re,¹⁸⁸Re, ¹⁸⁹Re, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ¹¹¹In, ¹¹¹Ag, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb,¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁹⁸Au, ²¹¹At, ²¹²Pb, ²²Bi, ²¹³Bi, ²²³Ra and ²²⁵Ac,and combinations thereof. Exemplary photoactive therapeutic agents areselected from the group comprising chromogens and dyes.

Still preferred, the therapeutic agent is an enzyme selected from thegroup comprising malate dehydrogenase, staphylococcal nuclease,delta-V-steroid isomerase, yeast alcohol dehydrogenase,α-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, β-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

The multispecific antagonist may bind specifically to at least oneproinflammatory effector cytokine, proinflammatory effector chemokine,or proinflammatory effector receptor. Proinflammatory effector cytokinesto which the multispecific antagonist may bind include, but are notrestricted to, MIF, HMGB-1, TNF-α (tumor necrosis factor alpha), IL-1,IL-4, IL-5, IL-6, IL-8, IL-12, IL-15, IL-17 and IL-18. Proinflammatoryeffector chemokines include, but are not restricted to, CCL19, CCL21,IL-8, MCP-1 (monocyte chemotactic protein 1), RANTES, MIP-1A (macrophageinflammatory protein 1A), MIP-1B (macrophage inflammatory protein 1B),ENA-78 (epithelial neutrophil activating peptide 78), IP-10, GROB (GRObeta), and Eotaxin. Proinflammatory effector receptors include, but arenot restricted to, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R and IL-18R. Themulti specific antagonist also may react specifically with at least onecoagulation factor, such as tissue factor or thrombin. Thelymphokines/cytokines react with their receptors on the immune cells toeffect activation, and antibodies can block activation by neutralizingthe lymphokine/cytokine. Alternatively, antibodies can react with thelymphokine/cytokine receptors to block activation.

The different targets to which the multispecific antagonist bindsspecifically may be from the same or different classes of effectors andcoagulation factors. For example, the two or more different targets towhich the antagonist binds specifically may be selected from the sameclass of effectors or coagulation factors, such as two or more differentproinflammatory effector cytokines, two ore more differentproinflammatory effector chemokines, two or more differentproinflammatory effector receptors, or two or more coagulation factors.Alternatively, the two or more different targets may be selected fromdifferent classes of effectors and coagulation factors. For example, onetarget may be a proinflammatory effector of the innate immune system andone target may be a coagulation factor. Or the antagonist may reactspecifically with two different classes of proinflammatory effectors,such as at least one proinflammatory effector cytokine and at least oneproinflammatory effector chemokine, at least one proinflammatoryeffector cytokine and at least one proinflammatory effector receptor, orat least one proinflammatory effector chemokine and at least oneproinflammatory effector receptor. It may also be the case that the twodifferent targets with which the multispecific antagonist reactsspecifically are more than one epitope of the same proinflammatoryeffector of the innate immune system or more than one epitope of thesame coagulation factor.

Thus, “two different targets” can refer to two different antigens, or totwo different epitopes of the same antigen. Multiple antibodies may beused against the same antigen, thus increasing valency. For example,when targeting MIF or HMGB-1, particularly for the treatment of sepsis,some cancers, and atherosclerotic plaques, two antibodies binding to twoidentical epitopes of the targets can be fused with another antibodyhaving one or more binding arms to a different antigen, such as an HLAclass II invariant chain antigen, such as CD74. These are examples ofbispecific or bifunctional antibodies that bind to two differentantigens, e.g., antibodies to MIF and CD74; antibodies to HMGB-1 andCD74. Trispecific and multispecific fusion proteins can also be made andused, thus targeting more than two antigens or receptor molecules. Thesecan have a single binding arm to each antigen or epitope, or more thanone, thus resulting in multivalency.

When a proinflammatory effector receptor is targeted, in a preferredembodiment the actual target may be an extracellular domain of theproinflammatory effector receptor. This extracellular domain of theproinflammatory effector receptor may be fused to an antibody. Moreparticularly, the proinflammatory effector may be a soluble receptor orreceptor ligand which is fused to an antibody. In an alternativeembodiment, the multispecific antagonist may comprise at least onemolecule reactive with a proinflammatory effector receptor. Thismolecule may be a natural antagonist for said proinflammatory effectorreceptor, or a fragment or mutant of this antagonist that interactsspecifically with the receptor. In a preferred embodiment, the naturalantagonist is the natural IL-1 receptor antagonist, or a fragment ormutant of this antagonist.

One of the at least two different targets to which the multispecificantagonist binds specifically may be a target that is not aproinflammatory effector of the immune system or a coagulation factor.In this case the multispecific antagonist also binds specifically withat least one proinflammatory effector of the immune system or at leastone coagulation factor. In one embodiment, this at least one othertarget is an antigen or receptor of the adaptive immune system. In otherembodiments, the at least one other target of the multispecificantagonist targets cells of the innate immune system, such asgranulocytes, monocytes, macrophages, dendritic cells, and NK-cells.Other targets include platelets and endothelial cells. Yet another groupof targets is the group consisting of C5a, LPS, IFNγ and B7. A furthergroup of suitable targets include CD2, CD4, CD14, CD18, CD11a, CD20,CD22, CD23, CD25, CD29, CD38, CD40L, CD52, CD64, CD83, CD147, and CD154.The CDs are targets on immune cells, which can be blocked by antibodiesto prevent an immune cell response. CD83 is particularly useful as amarker of activated dendritic cells (Cao et al., Biochem J., Aug. 23,2004 (Epub ahead of print); Zinser et al., J. Exp Med. 200(3):345-51(2004)).

Certain targets are of particular interest, such as MIF, HMGB-1, TNF-α,the complement factors and complement regulatory proteins, and thecoagulation factors. MIF is a pivotal cytokine in of the innate immunesystem and plays an important part in the control of inflammatoryresponses. Originally described as a T lymphocyte-derived factor thatinhibited the random migration of macrophages, the protein known asmacrophage migration inhibitory factor (MIF) was an enigmatic cytokinefor almost 3 decades. In recent years, the discovery of MIF as a productof the anterior pituitary gland and the cloning and expression ofbioactive, recombinant MIF protein have led to the definition of itscritical biological role in vivo. MIF has the unique property of beingreleased from macrophages and T lymphocytes that have been stimulated byglucocorticoids. Once released, MIF overcomes the inhibitory effects ofglucocorticoids on TNF-α, IL-1 beta, IL-6, and IL-8 production byLPS-stimulated monocytes in vitro and suppresses the protective effectsof steroids against lethal endotoxemia in vivo. MIF also antagonizesglucocorticoid inhibition of T-cell proliferation in vitro by restoringIL-2 and IFN-gamma production. MIF is the first mediator to beidentified that can counter-regulate the inhibitory effects ofglucocorticoids and thus plays a critical role in the host control ofinflammation and immunity. MIF is particularly useful in treatingcancer, pathological angiogenesis, and sepsis or septic shock.

HMGB-1, a DNA binding nuclear and cytosolic protein, is aproinflammatory cytokine released by monocytes and macrophages that havebeen activated by IL-1β, TNF, or LPS. Via its B box domain, it inducesphenotypic maturation of DCs. It also causes increased secretion of theproinflammatory cytokines IL-1 alpha, IL-6, IL-8, IL-12, TNF-α andRANTES. HMGB-1 released by necrotic cells may be a signal of tissue orcellular injury that, when sensed by DCs, induces and or enhances animmune reaction. Palumbo et al. report that HMBG1 induces mesoangioblastmigration and proliferation (J Cell Biol, 164:441-449 (2004)).

HMGB-1 is a late mediator of endotoxin-induced lethality that exhibitssignificantly delayed kinetics relate to TNF and IL-1beta. Experimentaltherapeutics that target specific early inflammatory mediators such asTNF and IL-1beta alone have not proven efficacious in the clinic, butmultispecific antagonists according to the present invention can improveresponse by targeting both early and late inflammatory inflammatorymediators.

Multispecific antagonists that target HMBG-1 are especially useful intreating arthritis, particularly collagen-induced arthritis.Multispecific antagonists comprising HMBG-1 also are useful in treatingsepsis and/or septic shock. Yang et al., PNAS USA 101:296-301 (2004);Kokkola et al., Arthritis Rheum, 48:2052-8 (2003); Czura et al., JInfect Dis, 187 Suppl 2:S391-6 (2003); Treutiger et al., J Intern Med,254:375-85 (2003).

TNF-α is an important cytokine involved in systemic inflammation and theacute phase response. TNF-α is released by stimulated monocytes,fibroblasts, and endothelial cells. Macrophages, T-cells andB-lymphocytes, granulocytes, smooth muscle cells, eosinophils,chondrocytes, osteoblasts, mast cells, glial cells, and keratinocytesalso produce TNF-α after stimulation. Its release is stimulated byseveral other mediators, such as interleukin-1 and bacterial endotoxin,in the course of damage, e.g., by infection. It has a number of actionson various organ systems, generally together with interleukins-1 and -6.One of the actions of TNF-α is appetite suppression; hence multispecificantagonists for treating cachexia preferably target TNF-α. It alsostimulates the acute phase response of the liver, leading to an increasein C-reactive protein and a number of other mediators. It also is auseful target when treating sepsis or septic shock.

The complement system is a complex cascade involving proteolyticcleavage of serum glycoproteins often activated by cell receptors. The“complement cascade” is constitutive and non-specific but it must beactivated in order to function. Complement activation results in aunidirectional sequence of enzymatic and biochemical reactions. In thiscascade, a specific complement protein, C5, forms two highly active,inflammatory byproducts, C5a and C5b, which jointly activate white bloodcells. This in turn evokes a number of other inflammatory byproducts,including injurious cytokines, inflammatory enzymes, and cell adhesionmolecules. Together, these byproducts can lead to the destruction oftissue seen in many inflammatory diseases. This cascade ultimatelyresults in induction of the inflammatory response, phagocyte chemotaxisand opsonization, and cell lysis.

The complement system can be activated via two distinct pathways, theclassical pathway and the alternate pathway. Most of the complementcomponents are numbered (e.g., C1, C2, C3, etc.) but some are referredto as “Factors.” Some of the components must be enzymatically cleaved toactivate their function; others simply combine to form complexes thatare active. Active components of the classical pathway include C1q, C1r,C1s, C2a, C2b, C3a, C3b, C4a, and C4b. Active components of thealternate pathway include C3a, C3b, Factor B, Factor Ba, Factor Bb,Factor D, and Properdin. The last stage of each pathway is the same, andinvolves component assembly into a membrane attack complex. Activecomponents of the membrane attack complex include C5a, C5b, C6, C7, C8,and C9n.

While any of these components of the complement system can be targetedby a multispecific antagonist according to the invention, certain of thecomplement components are preferred. C3a, C4a and C5a cause mast cellsto release chemotactic factors such as histamine and serotonin, whichattract phagocytes, antibodies and complement, etc. These form one groupof preferred targets according to the invention. Another group ofpreferred targets includes C3b, C4b and C5b, which enhance phagocytosisof foreign cells. Another preferred group of targets are the predecessorcomponents for these two groups, i.e., C3, C4 and C5. C5b, C6, C7, C8and C9 induce lysis of foreign cells (membrane attack complex) and formyet another preferred group of targets.

Complement C5a, like C3a, is an anaphylatoxin. It mediates inflammationand is a chemotactic attractant for induction of neutrophilic release ofantimicrobial proteases and oxygen radicals. Therefore, C5a and itspredecessor C5 are particularly preferred targets. By targeting C5, notonly is C5a affected, but also C5b, which initiates assembly of themembrane-attack complex. Thus, C5 is another preferred target. C3b, andits predecessor C3, also are preferred targets, as both the classicaland alternate complement pathways depend upon C3b. Three proteins affectthe levels of this factor, C1 inhibitor, protein H and Factor I, andthese are also preferred targets according to the invention. Complementregulatory proteins, such as CD46, CD55, and CD59, may be targets towhich the multispecific antagonists bind.

Coagulation factors also are preferred targets according to theinvention, particularly tissue factor (TF) and thrombin. TF is alsoknown also as tissue thromboplastin, CD142, coagulation factor III, orfactor III. TF is an integral membrane receptor glycoprotein and amember of the cytokine receptor superfamily. The ligand bindingextracellular domain of TF consists of two structural modules withfeatures that are consistent with the classification of TF as a memberof type-2 cytokine receptors. TF is involved in the blood coagulationprotease cascade and initiates both the extrinsic and intrinsic bloodcoagulation cascades by forming high affinity complexes between theextracellular domain of TF and the circulating blood coagulationfactors, serine proteases factor VII or factor VIIa. These enzymaticallyactive complexes then activate factor IX and factor X, leading tothrombin generation and clot formation.

TF is expressed by various cell types, including monocytes, macrophagesand vascular endothelial cells, and is induced by IL-1, TNF-α orbacterial lipopolysaccharides. Protein kinase C is involved in cytokineactivation of endothelial cell TF expression. Induction of TF byendotoxin and cytokines is an important mechanism for initiation ofdisseminated intravascular coagulation seen in patients withGram-negative sepsis. TF also appears to be involved in a variety ofnon-hemostatic functions including inflammation, cancer, brain function,immune response, and tumor-associated angiogenesis. Thus, multispecificantagonists that target TF are useful not only in the treatment ofcoagulopathies, but also in the treatment of sepsis, cancer, pathologicangiogenesis, and other immune and inflammatory dysregulatory diseasesaccording to the invention. A complex interaction between thecoagulation pathway and the cytokine network is suggested by the abilityof several cytokines to influence TF expression in a variety of cellsand by the effects of ligand binding to the receptor. Ligand binding(factor VIIa) has been reported to give an intracellular calcium signal,thus indicating that TF is a true receptor.

Thrombin is the activated form of coagulation factor II (prothrombin);it converts fibrinogen to fibrin. Thrombin is a potent chemotaxin formacrophages, and can alter their production of cytokines and arachidonicacid metabolites. It is of particular importance in the coagulopathiesthat accompany sepsis. Numerous studies have documented the activationof the coagulation system either in septic patients or following LPSadministration in animal models. Despite more than thirty years ofresearch, the mechanisms of LPS-induced liver toxicity remain poorlyunderstood. It is now clear that they involve a complex and sequentialseries of interactions between cellular and humoral mediators. In thesame period of time, gram-negative systemic sepsis and its sequallaehave become a major health concern, attempts to use monoclonalantibodies directed against LPS or various inflammatory mediators haveyielded only therapeutic failures, as noted elsewhere herein.Multispecific antagonists according to the invention that target boththrombin and at least one other target address the clinical failures insepsis treatment.

In other embodiments, the multispecific antagonists bind to a MHC classI, MHC class II or accessory molecule, such as CD40, CD54, CD80 or CD86.The multispecific antagonist also may bind to a T-cell activationcytokine, or to a cytokine mediator, such as NF-κB.

In certain embodiments, one of the at least two different targets may bea cancer cell receptor or cancer-associated antigen, particularly onethat is selected from the group consisting of B-cell lineage antigens(CD19, CD20, CD21, CD22, CD23, etc.), VEGFR, EGFR, carcinoembryonicantigen (CEA), placental growth factor (PLGF), tenascin, HER-2/neu,EGP-1, EGP-2, CD25, CD30, CD33, CD38, CD40, CD45, CD52, CD74, CD80,CD138, NCA66, CEACAM6 (carcinoembryonic antigen-related cellularadhesion molecule 6), MUC1, MUC2, MUC3, MUC4, MUC16, IL-6, α-fetoprotein(AFP), A3, CA125, colon-specific antigen-p (CSAp), folate receptor,HLA-DR, human chorionic gonadotropin (HCG), Ia, EL-2, insulin-likegrowth factor (ILGF) and ILGF receptor, KS-1, Le(y), MAGE, necrosisantigens, PAM-4, prostatic acid phosphatase (PAP), Pr1, prostatespecific antigen (PSA), prostate specific membrane antigen (PSMA), S100,T101, TAC, TAG72, TRAIL receptors, and carbonic anhydrase IX.

Targets associated with sepsis and immune dysregulation and other immunedisorders include MIF, IL-1, IL-6, IL-8, CD74, CD83, and C5aR.Antibodies and inhibitors against C5aR have been found to improvesurvival in rodents with sepsis (Huber-Lang et al., FASEB J 2002;16:1567-1574; Riedemann et al., J Clin Invest 2002; 110: 101-108) andseptic shock and adult respiratory distress syndrome in monkeys (Hangenet al., J Surg Res 1989; 46:195-199; Stevens et al., J Clin Invest 1986;77:1812-1816). Thus, for sepsis, one of the at least two differenttargets preferably is a target that is associated with infection, suchas LPS/C5a. Other preferred targets include HMGB-1, TF, CD14, VEGF, andIL-6, each of which is associated with septicemia or septic shock.Preferred multispecific antagonists are those that target two or moretargets from HMGB-1, TF and MIF, such as MIF/TF, and HMGB-1/TF.

In still other embodiments, one of the at least two different targetsmay be a target this is associated with graft versus host disease ortransplant rejection, such as MIF (Lo et al., Bone Marrow Transplant,30(6):375-80 (2002)). One of the at least two different targets also mayone that associated with acute respiratory distress syndrome, such asIL-8 (Bouros et al., PMC Pulm Med, 4(1):6 (2004), atherosclerosis orrestenosis, such as MIF (Chen et al., Arterioscler Thromb Vasc Biol,24(4):709-14 (2004), asthma, such as IL-18 (Hata et al., Int Immunol,Oct. 11, 2004 Epub ahead of print), a granulomatous disease, such asTNF-α (Ulbricht et al., Arthritis Rheum, 50(8):2717-8 (2004), aneuropathy, such as carbamylated EPO (erythropoietin) (Leist et al.,Science 305(5681):164-5 (2004), or cachexia, such as IL-6 and TNF-α.

Other targets include C5a, LPS, IFN-gamma, B7; CD2, CD4, CD14, CD18,CD11a, CD11b, CD11c, CD14, CD18, CD27, CD29, CD38, CD40L, CD52, CD64,CD83, CD147, CD154. Activation of mononuclear cells by certain microbialantigens, including LPS, can be inhibited to some extent by antibodiesto CD18, CD11b, or CD11c, which thus implicate β₂-integrins (Cuzzola etal., J Immunol 2000; 164:5871-5876; Medvedev et al., J Immunol 1998;160: 4535-4542). CD83 has been found to play a role in giant cellarteritis (GCA), which is a systemic vasculitis that affects medium- andlarge-size arteries, predominately the extracranial branches of theaortic arch and of the aorta itself, resulting in vascular stenosis andsubsequent tissue ischemia, and the severe complications of blindness,stroke and aortic arch syndrome (Weyand and Goronzy, N Engl J Med 2003;349:160-169; Hunder and Valente, In: Inflammatory Diseases of BloodVessels. G. S. Hoffman and C. M. Weyand, eds, Marcel Dekker, New York,2002; 255-265). Antibodies to CD83 were found to abrogate vasculitis ina SCID mouse model of human GCA (Ma-Krupa et al., J Exp Med 2004;199:173-183), suggesting to these investigators that dendritic cells,which express CD83 when activated, are critical antigen-processing cellsin GCA. In these studies, they used a mouse anti-CD83 Mab (IgG1 cloneHB15e from Research Diagnostics). CD154, a member of the TNF family, isexpressed on the surface of CD4-positive T-lymphocytes, and it has beenreported that a humanized monoclonal antibody to CD154 producedsignificant clinical benefit in patients with active systemic lupuserythematosus (SLE) (Grammar et al., J Clin Invest 2003; 112:1506-1520).It also suggests that this antibody might be useful in other autoimmunediseases (Kelsoe, J Clin Invest 2003; 112:1480-1482). Indeed, thisantibody was also reported as effective in patients with refractoryimmune thrombocytopenic purpura (Kuwana et al., Blood 2004;103:1229-1236).

In rheumatoid arthritis, a recombinant interleukin-1 receptorantagonist, IL-1Ra or anakinra (Kineret®), has shown activity (Cohen etal., Ann Rheum Dis 2004; 63:1062-8; Cohen, Rheum Dis Clin North Am 2004;30:365-80). An improvement in treatment of these patients, whichhitherto required concomitant treatment with methotrexate, is to combineanakinra with one or more of the anti-proinflammatory effector cytokinesor anti-proinflammatory effector chemokines (as listed above). Indeed,in a review of antibody therapy for rheumatoid arthritis, Taylor (CurrOpin Pharmacol 2003; 3:323-328) suggests that in addition to TNF, otherantibodies to such cytokines as IL-1, IL-6, IL-8, IL-15, IL-17 andIL-18, are useful.

Some of the more preferred target combinations include the following:

First target Second target MIF A second proinflammatory effectorcytokine, especially HMGB-1, TNF-α, IL-1, or IL-6 MIF Proinflammatoryeffector chemokine, especially MCP-1, RANTES, MIP-1A, or MIP-1B MIFProinflammatory effector receptor, especially IL-6R IL-13R, and IL-15RMIF Coagulation factor, especially TF or thrombin MIF Complement factor,especially C3, C5, C3a, or C5a MIF Complement regulatory protein,especially CD46, CD55, CD59, and mCRP MIF Cancer associated antigen orreceptor HMGB-1 A second proinflammatory effector cytokine, especiallyMIF, TNF-α, IL-1, or IL-6 HMGB-1 Proinflammatory effector chemokine,especially MCP-1, RANTES, MIP-1A, or MIP-1B HMGB-1 Proinflammatoryeffector receptor especially MCP-1, RANTES, MIP-1A, or MIP-1B HMGB-1Coagulation factor, especially TF or thrombin HMGB-1 Complement factor,especially C3, C5, C3a, or C5a HMGB-1 Complement regulatory protein,especially CD46, CD55, CD59, and mCRP HMGB-1 Cancer associated antigenor receptor TNF-α A second proinflammatory effector cytokine, especiallyMIF, HMGB-1, TNF-α, IL-1, or IL-6 TNF-α Proinflammatory effectorchemokine, especially MCP-1, RANTES, MIP-1A, or MIP-1B TNF-αProinflammatory effector receptor, especially IL-6R IL-13R, and IL-15RTNF-α Coagulation factor, especially TF or thrombin TNF-α Complementfactor, especially C3, C5, C3a, or C5a TNF-α Complement regulatoryprotein, especially CD46, CD55, CD59, and mCRP TNF-α Cancer associatedantigen or receptor LPS Proinflammatory effector cytokine, especiallyMIF, HMGB-1, TNF-α, IL-1, or IL-6 LPS Proinflammatory effectorchemokine, especially MCP-1, RANTES, MIP-1A, or MIP-1B LPSProinflammatory effector receptor, especially IL-6R IL-13R, and IL-15RLPS Coagulation factor, especially TF or thrombin LPS Complement factor,especially C3, C5, C3a, or C5a LPS Complement regulatory protein,especially CD46, CD55, CD59, and mCRP TF or Proinflammatory effectorcytokine, especially MIF, thrombin HMGB-1, TNF-α, IL-1, or IL-6 TF orProinflammatory effector chemokine, especially MCP-1, thrombin RANTES,MIP-1A, or MIP-1B TF or Proinflammatory effector receptor, especiallyIL-6R thrombin IL-13R, and IL-15R TF or Complement factor, especiallyC3, C5, C3a, or C5a thrombin TF or Complement regulatory protein,especially CD46, CD55, thrombin CD59, and mCRP TF or Cancer associatedantigen or receptor thrombinIn each of the above, the multispecific may include additional targets,e.g., third and further targets. This is a list of examples of preferredcombinations, but is not intended to be exhaustive.

While the multispecific antagonist may be a mixture that contains atleast two separate antibodies and/or receptors or their ligands thatbind to the different targets, in one preferred embodiment theantagonist is an antibody that is at least bispecific, in whichdifferent arms of the antibody react specifically with at least twodifferent targets, wherein the targets are selected from the groupconsisting of proinflammatory effectors of the innate immune system,coagulation factors, complement factors and complement regulatoryproteins, and targets specifically associated with an inflammatory orimmune-dysregulatory disorder, with a pathologic angiogenesis or cancer,or with an infectious disease.

There are certain advantages when the multispecific antagonist is anantibody that is at least bispecific, including rapid clearance from theblood. For example, the bispecific antibody may bind to a receptor or toits target molecule, such as for LPS, IL-1, IL-10, IL-6, MIF, HMGB1,TNF, IFN, tissue factor, thrombin, CD14, CD27, and CD134. Many of theseexist as both receptors and as soluble forms in the blood. Binding bythe bispecific antibodies results in rapid clearance from the blood, andthen targeting by the second arm of the fusion protein to another cell,such as a macrophage, for transport and degradation by the cell,especially the lysosomes. This is particularly effective when the secondtargeting arm is against an internalizing antigen, such as CD74,expressed by macrophages and dendritic cells. This is consistent withthe invention of Hansen, U.S. Pat. No. 6,458,933, but focusing herein oninflammatory cytokines and other immune modulation molecules andreceptors for immune-dysregulation diseases, and cancer antigens for theimmunotherapy of these cancers.

The multispecific antagonist may contain a single active component, orit may contain multiple active components. For example, when themultispecific antagonists comprise multiple separate antibodies, theseconstitute multiple active components. Alternatively, the multispecificantagonist may be a single active component, such as a multispecificantibody that reacts specifically with at least two different targets ora monospecific or multispecific antibody fused to a soluble receptor.The multispecific antagonist also may be packaged together with othersecondary therapeutic modalities which are described below. The activecomponents may be packaged together with one or more inactivecomponents, such as a carrier or diluent, with instructions explainingthe manner in which the components are to be combined. All componentsare conveniently packaged together in kit form with instructionsregarding the combination and administration of the kit components. Thecomponents and agents may also be packaged and supplied separately.

Preferred multispecific antagonists for the treatment of cancer includeantibodies to CD55 and to any of the above cancer antigens, antibodiesto CD46 and to any of the above cancer antigens, antibodies to CD59 andto any of the above cancer antigens, antibodies to MIF and to any of theabove cancer antigens, antibodies to NF-κB and any of the above cancerantigens, and antibodies to IL-6 and to any of the above cancer antigensother than IL-6. These multispecific antagonists for treating cancer maybe antibody combinations or fusion proteins, given together orseparately.

The multispecific antagonist may be used in conjunction with one or moresecondary therapeutics. This secondary therapeutic may be one thataffects a component of the innate immune system. Alternatively, it mayaffect a component of the adaptive immune system. The secondarytherapeutic may also be a component that affects coagulation, cancer, oran autoimmune disease, such as a cytotoxic drug.

The multispecific antagonist with a diagnostic or therapeutic agent maybe provided as a kit for human or mammalian therapeutic and diagnosticuse in a pharmaceutically acceptable injection vehicle, preferablyphosphate-buffered saline (PBS) at physiological pH and concentration.The preparation preferably will be sterile, especially if it is intendedfor use in humans. Optional components of such kits include stabilizers,buffers, labeling reagents, radioisotopes, paramagnetic compounds,second antibody for enhanced clearance, and conventional syringes,columns, vials and the like.

Production of Monoclonal Antibodies, Humanized Antibodies, PrimateAntibodies and Human Antibodies

Rodent monoclonal antibodies to available antigens can be obtained bymethods known to those skilled in the art. See generally, for example,Kohler and Milstein, Nature 256:495 (1975), and Coligan et al. (eds.),CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley &Sons 1991) [“Coligan”]. Briefly, monoclonal antibodies can be obtainedby injecting mice with a composition comprising the antigen, verifyingthe presence of antibody production by removing a serum sample, removingthe spleen to obtain B-lymphocytes, fusing the B-lymphocytes withmyeloma cells to produce hybridomas, cloning the hybridomas, selectingpositive clones which produce antibodies to the antigen that wasinjected, culturing the clones that produce antibodies to the antigen,and isolating the antibodies from the hybridoma cultures.

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, seeBaines et al., “Purification of Immunoglobulin G (IgG),” in METHODS INMOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

Suitable amounts of well-characterized antigen for production ofantibodies can be obtained using standard techniques. As an example,CD22 can be immunoprecipitated from B-lymphocyte protein using thedeposited antibodies described by Tedder et al., U.S. Pat. No. 5,484,892(1996). Alternatively, antigen proteins can be obtained from transfectedcultured cells that overproduce the antigen of interest. Expressionvectors that comprise DNA molecules encoding each of these proteins canbe constructed using published nucleotide sequences. See, for example,Wilson et al., J. Exp. Med. 173:137 (1991); Wilson et al., J. Immunol.150:5013 (1993). DNA molecules encoding the antigen of interest can beobtained by synthesizing DNA molecules using mutually priming longoligonucleotides. See, for example, Ausubel et al., (eds.), CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, pages 8.2.8 to 8.2.13 (1990)[“Ausubel”]. Also, see Wosnick et al., Gene 60:115 (1987); and Ausubelet al. (eds.), SHORT PROTOCOLS IN MOLECULAR BIOLOGY, 3rd Edition, pages8-8 to 8-9 (John Wiley & Sons, Inc. 1995). Established techniques usingthe polymerase chain reaction provide the ability to synthesize genes aslarge as 1.8 kilobases in length. Adang et al., Plant Molec. Biol.21:1131 (1993); Bambot et al., PCR Methods and Applications 2:266(1993); Dillon et al., “Use of the Polymerase Chain Reaction for theRapid Construction of Synthetic Genes,” in METHODS IN MOLECULAR BIOLOGY,Vol. 15: PCR PROTOCOLS: CURRENT METHODS AND APPLICATIONS, White (ed.),pages 263-268, (Humana Press, Inc. 1993).

In an alternative embodiment, an antibody of the present invention is achimeric antibody in which the variable regions of a human antibody havebeen replaced by the variable regions of a rodent antibody. Theadvantages of chimeric antibodies include decreased immunogenicity andincreased in vivo stability.

Techniques for constructing chimeric antibodies are well known to thoseof skill in the art. As an example, Leung et al., Hybridoma 13:469(1994), describe how they produced an LL2 chimera by combining DNAsequences encoding the V_(κ) and V_(H) domains of LL2 monoclonalantibody with respective human κ and IgG₁ constant region domains. Thispublication also provides the nucleotide sequences of the LL2 light andheavy chain variable regions, V_(κ) and V_(H), respectively.

In another embodiment, an antibody of the present invention is asubhuman primate antibody. General techniques for raisingtherapeutically useful antibodies in baboons may be found, for example,in Goldenberg et al., international patent publication No. WO 91/11465(1991), and in Losman et al., Int J Cancer 46: 310 (1990).

In yet another embodiment, an antibody of the present invention is a“humanized” monoclonal antibody. That is, mouse complementaritydetermining regions (CDRs) are transferred from heavy and light variablechains of the mouse immunoglobulin into a human variable domain,followed by the replacement of some human residues in the frameworkregions of their murine counterparts. Humanized monoclonal antibodies inaccordance with this invention are suitable for use in therapeuticmethods. General techniques for cloning murine immunoglobulin variabledomains are described, for example, by the publication of Orlandi etal., Proc Nat'l Acad Sci USA 86:3833 (1989). Techniques for producinghumanized monoclonal antibodies are described, for example, by Jones etal., Nature 321:522 (1986), Riechmann et al., Nature 332:323 (1988),Verhoeyen et al., Science 239:1534 (1988), Carter et al., Proc. Nat'lAcad. Sci. USA 89:4285 (1992), Sandhu, Crit Rev Biotech. 12:437 (1992),and Singer et al., J Immun 150:2844 (1993). The publication of Leung etal., Mol Immunol 32:1413 (1995), describes the construction of humanizedLL2 antibody.

In another embodiment, an antibody of the present invention is a humanmonoclonal antibody. Such antibodies are obtained from transgenic micethat have been “engineered” to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain locus are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous heavy chain and light chain loci. The transgenic micecan synthesize human antibodies specific for human antigens, and themice can be used to produce human antibody-secreting hybridomas. Methodsfor obtaining human antibodies from transgenic mice are described byGreen et al., Nature Genet 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int Immun 6:579 (1994).

A fully human antibody also can be constructed by genetic or chromosomaltransfection methods, as well as phage display technology, all of whichare known in the art. See for example, McCafferty et al., Nature348:552-553 (1990) for the production of human antibodies and fragmentsthereof in vitro, from immunoglobulin variable domain gene repertoiresfrom unimmunized donors. In this technique, antibody variable domaingenes are cloned in-frame into either a major or minor coat protein geneof a filamentous bacteriophage, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. In this way, the phage mimics some of the properties of theB cell. Phage display can be performed in a variety of formats, fortheir review, see e.g., Johnson and Chiswell, Current Opinion inStructural Biology, 3:5564-571 (1993).

Although xenogeneic antibodies may be used in the invention, it ispreferable to use allogeneic antibodies to reduce the likelihood of theantibodies themselves inducing an immune response from the host. In aparticular embodiment of the invention, a human antibody is used.Methods for making fully human antibodies for use in human subjectsinclude the use of phage display techniques for selecting antigenspecific antibodies from a large human antibody library, as described inU.S. Pat. No. 5,969,108, which is incorporated herein by reference inits entirety. Other phage display methods for making human antibodiesfrom designed human antibody libraries are described in U.S. Pat. No.6,300,064, which is incorporated herein by reference in its entirety.See also: Marks, et al. “By-Passing Immunization: Building High AffinityHuman Antibodies by Chain Shuffling.” (Bio/Technology, vol. 10: p.779-783. (1992)), Hoogenboom, et al. “Building Antibodies From TheirGenes.” (Rev Fr Transfus Hemobiol, vol. 36: p. 19-47, (1993));Griffiths, et al. “Isolation of High Affinity Human Antibodies Directlyfrom Large Synthetic Repertoires.” EMBO J, vol. 13: p. 3245-3260(1994)); Winter and Milstein “Man-Made Antibodies.” (Nature, vol. 349:p. 293-299 (1991)); De Kruif, et al., “Selection and Application ofHuman Single Chain Fv Antibody Fragments from a Semi-synthetic PhageAntibody Display Library with Designed CDR3 Regions,” (J Mol Biol, vol.248, pp. 97-105 (1995)) and Barbas et al., “Semisynthetic combinatorialantibody libraries: A chemical solution to the diversity problem,” (ProcNatl Acad Sci USA, vol. 89, pp. 4457-4461 (1992)). Other methods formaking fully human antibodies include the use of so-called “xenomouse”technology, using transgenic mice that encode a large portion of thehuman antibody repertoire. These methods are provided commercially by,for example, Abgenix (Fremont Calif.) and Medarex (Princeton N.J.). Seealso, U.S. Pat. No. 6,075,181; Lonberg, “Transgenic Approaches to HumanMonoclonal Antibodies.” Handbook of Experimental Pharmacology 113(1994): 49-101; Lonberg et al., “Human Antibodies from Transgenic Mice.”Internal Review of Immunology 13 (1995): 65-93.

Production of Multispecific Antibodies

The multispecific antagonists according to the present invention may bemultispecific antibodies or antibody fragments, particularly bispecificantibodies (bsAb) or bispecfic antibody fragments (bsFab). Thesemultispecific antagonists have arms that specifically bind to at leasttwo different targets, where the targets are selected from the groupconsisting of proinflammatory effectors of the innate immune system,coagulation factors, and targets specifically associated with aninflammatory or immune-dysregulatory disorder or with a pathologicangiogenesis, where the latter target is not a proinflammatory effectorof the immune system, a coagulation factor or a cancer cell receptor orcancer-associated antigen.

The present invention may employ a pretargeting strategy, in which onearm of a multispecific antibody or fragment binds to a targetableconjugate. Pretargeting strategies based on avidin or streptavidin andbiotin may be used, as described in Goldenberg, U.S. Pat. No. 5,525,338,entitled “Detection and Therapy of Lesions with Biotin/AvidinConjugates.” The avidin/streptavidin system is highly versatile and hasbeen used in several configurations. Antibodies can be coupled withstreptavidin or biotin, which is used as the primary targeting agent.This is followed sometime later by the therapeutic agent, which isconjugated with biotin or with avidin/streptavidin, respectively.Another configuration relies on a 3-step approach first targeting abiotin-conjugated antibody, followed by a bridging withstreptavidin/avidin, and then the biotin-conjugated therapeutic agent isgiven. Description of biotin and avidin/streptavidin conjugation toantibodies and other species is well-known in the art. See, for example,Griffiths et al., U.S. Pat. No. 5,846,741; Griffiths et al, U.S. Pat.No. 5,965,115, and Griffiths et al., U.S. Pat. No. 6,120,768. While theavidin-biotin system has a very high affinity, clinical experience hasshown that approximately 20-30% of patients mount an antibody responseagainst avidin and up to 70% make antibodies to streptavidin.Accordingly, low-molecular weight haptens are more preferred.

In this embodiment, the multispecific antagonist is a multispecificantibody that comprises an arm that is specific for a low-molecularweight hapten to which a therapeutic agent is conjugated or fused. Inthis case, the antibody pretargets the cells, and the low-molecularweight hapten with the attached therapeutic agent is administered afterthe antibody has bound to the targets. Examples of recognizable haptensinclude, but are not limited to, chelators, such as DTPA, DOTA,fluorescein isothiocyanate, vitamin B-12 and other moieties to whichspecific antibodies can be raised. The subsequently injected haptens cancarry different diagnostic or therapeutic agents. More than onemultispecific antibody also may be used, each of which comprises an armwhich recognizes the same hapten. The use of multi specific antibodies,and combinations of multi specific antibodies, is particularly effectivein overcoming antigen heterogeneity in tumors and other diseased tissue.

The use of hapten-therapy agent conjugates for the localization oftherapeutics to disease targets has several distinct advantages. Thesame hapten can be attached to several different therapy agents. Inaddition, should an immune response to one therapy agent be seen, thiswill not destroy the ability of a developed non-immunogenic antibodytargeting vector to be used in hapten-therapy agent-based systems. Thisalso enables the use of a universal targeting system that can be usedwith any targeting antibody and therapy agent combination. In using ahapten recognition system the designing chemist has control over howhaptens are attached to a therapy agent, and is able to incorporatefeatures such as lability to a particular extra-cellular orintracellular enzyme, or instability to a particular set of conditions,such as slightly lowered pH.

The carrier portion is conjugated to a proinflammatory effector of theinnate immune system, a coagulation factor, or a target specificallyassociated with an inflammatory or immune-dysregulatory disorder or witha pathologic angiogenesis that is neither a proinflammatory effector ofthe innate immune system or a coagulation factor. The use ofmultispecific antibodies and fragments which have at least one arm thatspecifically binds a targetable conjugate allows a variety oftherapeutic applications to be performed without raising newmultispecific antibody for each application.

MAbs can be raised to any hapten or drug by standard methods of makingmAbs known to a person skilled in the art. For instance, it is possibleto attach, a hapten such as HSG (histamine-succinyl-glycine) to animmunogenic stimulator or adjuvant such a keyhole limpet hemocyanin, andinject the conjugate into immunocompetent animals. Multiple injectionsare often employed. It must be appreciated that such an approach canlead to several different antibodies with slightly differentspecificities against the hapten in question, such as HSG. MAbs canrecognize different sub-parts of the HSG structure, or differentconformations. MAbs may also be obtained that recognize a little morethan just the HSG molecule itself, such as recognizing an HSG moietyonly when attached to an epsilon amino group of lysine, if indeed, theHSG was initially linked to the KLH (for example) by attachment to anepsilon lysyl amino group on the latter immunogenic protein. Withoutwishing to be exhaustive, these general procedures and results are wellknown in the art. It is also then well known art for the isolation ofspleen cells producing antibodies from these immunized animals, andtheir subsequent fusion with myeloma cell lines, to generate hybridomassecreting anti-hapten antibodies. See Kohler G. and Milstein C., Eur JImmunol 6:511-9 (1976); Kohler G. et al., Eur J Immunol 6:292-5 (1976);and Kohler G. and Milstein C., Nature 256:495-7 (1975).

Multispecific targeting proteins can be prepared chemically fromantibodies that have differing specificity by well-known reactions.Typically, one MAb is activated by reaction with a cross-linking agent,with the latter chosen to react at the first MAb's lysine, reducedcysteine, or oxidized carbohydrate residues. After purification, theactivated first MAb is mixed with the second MAb, which then reactsspecifically with a second functionality of the original cross-linkingagent; most notably via the second MAb's lysine, reduced cysteine oroxidized carbohydrate residues. Multispecific targeting proteins canalso be prepared somatically by the quadroma technique. The quadromatechnique is a technique wherein a cell line expressing both arms of thebispecific antibody is produced and grown in culture to secrete thebsMAb. Finally, bsMAbs can also be produced conveniently by moderntechniques of molecular biology. See, for example, Colman, A., BiochemSoc Symp 63: 141-147 (1998); U.S. Pat. No. 5,827,690; and Published U.S.Application 20020006379.

The present invention encompasses antibodies and antibody fragments.Antibodies are generally bivalent, or less often multivalent, and thisbivalency enhances the strength of attachment of the antibody to cellsurfaces. However, the bivalency of the antibody sometimes induces atarget cell to undergo antigenic modulation thereby providing a meanswhereby the cell can avoid the cytotoxic agents, effector cells andcomplement, which are involved in the cell-antibody interaction. As ameans of preventing such modulation, monovalent antibodies or antibodyfragments can be used. A monovalent antibody is a complete, functionalimmunoglobulin molecule in which only one of the light chains binds toantigen. One method of preparing such antibodies is disclosed in U.S.Pat. No. 4,841,025.

Monovalency can be achieved by using antibody fragments. Exemplarymonovalent antibody fragments useful in these embodiments are Fv, Fab,Fab′ and the like. Monovalent antibody fragments, typically exhibiting amolecular weight ranging from about 25 kD (Fv) to about 50 kD (Fab,Fab′), are smaller than whole antibody and, therefore, are generallycapable of greater target site penetration. Moreover, monovalent bindingcan result in less binding carrier restriction at the target surface(occurring during use of bivalent antibodies, which bind strongly andadhere to target cell sites thereby creating a barrier to further egressinto sublayers of target tissue), thereby improving the homogeneity oftargeting. In addition, smaller molecules are more rapidly cleared froma recipient, thereby decreasing the immunogenicity of the administeredsmall molecule conjugate. A lower percentage of the administered dose ofa monovalent fragment conjugate localizes to target in comparison to awhole antibody conjugate. The decreased immunogenicity may permit agreater initial dose of the monovalent fragment conjugate to beadministered, however. In addition, monovalent antibody fragmentsgenerally do not reside as long on the target cell as do bivalent orwhole antibodies.

The antibody fragments are antigen binding portions of an antibody, suchas F(ab′)2, F(ab)₂, Fab′, Fab, and the like. The antibody fragments bindto the same antigen that is recognized by the intact antibody. Forexample, an anti-CD22 monoclonal antibody fragment binds to an epitopeof CD22. The bsAb of the present invention include, but are not limitedto, IgG×IgG, IgG×F(ab′)₂, IgG×Fab′, IgG×scFv, F(ab′)₂×F(ab′)₂,Fab′×F(ab′)₂, Fab′×Fab′, Fab′×scFv and scFv×scFv bsMabs. Also, speciessuch as scFv×IgG×scFv and Fab′×IgG×Fab′, scFv×F(ab′)₂×scFv andFab′×F(ab′)₂×Fab′ are included.

As noted above, the term “antibody fragment” also includes any syntheticor genetically engineered protein that acts like an antibody by bindingto a specific antigen to form a complex. For example, antibody fragmentsinclude isolated fragments, “Fv” fragments, consisting of the variableregions of the heavy and light chains, recombinant single chainpolypeptide molecules in which light and heavy chain variable regionsare connected by a peptide linker (“sFv proteins”), and minimalrecognition units consisting of the amino acid residues that mimic thehypervariable region.

Production of Fusion Proteins

Another method for producing multispecific antagonists is to engineerrecombinant fusion proteins linking two or more different single-chainantibody or antibody fragment segments with the needed multiplespecificities. See, e.g., Coloma et al., Nature Biotech 15:159-163,1997. For example, a variety of bispecific fusion proteins can beproduced using molecular engineering. In one form, the bispecific fusionprotein is monovalent, consisting of, for example, a scFv with a singlebinding site for one antigen and a Fab fragment with a single bindingsite for a second antigen. In another form, the bispecific fusionprotein is divalent, consisting of, for example, an IgG with two bindingsites for one antigen and two scFv with two binding sites for a secondantigen.

Functional bispecific single-chain antibodies (bscAb), also calleddiabodies, can be produced in mammalian cells using recombinant methods.See, e.g., Mack et al., Proc Natl Acad Sci, 92:7021-7025, 1995. Forexample, bscAb are produced by joining two single-chain Fv fragments viaa glycine-serine linker using recombinant methods. The V light-chain(V_(L)) and V heavy-chain (V_(H)) domains of two antibodies of interestare isolated using standard PCR methods. The V_(L) and V_(H) cDNA'sobtained from each hybridoma are then joined to form a single-chainfragment in a two-step fusion PCR. The first PCR step introduces the(Gly4-Ser1)₃ linker, and the second step joins the V_(L) and V_(H)amplicons. Each single chain molecule is then cloned into a bacterialexpression vector. Following amplification, one of the single-chainmolecules is excised and sub-cloned into the other vector, containingthe second single-chain molecule of interest. The resulting bscAbfragment is subcloned into a eukaryotic expression vector. Functionalprotein expression can be obtained by transfecting the vector intoChinese hamster ovary cells. Recombinant methods can be used to producea variety of fusion proteins.

Production of Immunoconjugates

Any of the multispecific antagonists of the present invention can beconjugated with one or more therapeutic or diagnostic/detection agents.Generally, one therapeutic or diagnostic/detection agent is attached toeach antibody, fusion protein or fragment thereof but more than onetherapeutic agent and/or diagnostic/detection agent can be attached tothe same antibody or antibody fragment. If the Fc region is absent (forexample when the antibody used as the antibody component of theimmunoconjugate is an antibody fragment), it is possible to introduce acarbohydrate moiety into the light chain variable region of a fulllength antibody or antibody fragment. See, for example, Leung et al., JImmunol 154: 5919 (1995); Hansen et al., U.S. Pat. No. 5,443,953 (1995),Leung et al., U.S. Pat. No. 6,254,868. The engineered carbohydratemoiety is used to attach the therapeutic or diagnostic/detection agent.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int J Cancer 41: 832 (1988); Shih et al., IntJ Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No. 5,057,313. Thegeneral method involves reaction of an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function and that is loaded with a plurality of peptide.This reaction results in an initial Schiff base (imine) linkage, whichcan be stabilized by reduction to a secondary amine to form the finalconjugate.

A therapeutic or diagnostic/detection agent can be attached at the hingeregion of a reduced antibody component via disulfide bond formation. Asan alternative, such agents can be attached to the antibody componentusing a heterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int J Cancer 56: 244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION ANDCROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLESAND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). Alternatively, the therapeutic ordiagnostic/detection agent can be conjugated via a carbohydrate moietyin the Fc region of the antibody. The carbohydrate group can be used toincrease the loading of the same agent that is bound to a thiol group,or the carbohydrate moiety can be used to bind a different peptide.

Coupling of Antibodies to Lipid Emulsions

Long-circulating sub-micron lipid emulsions, stabilized withpoly(ethylene glycol)-modified phosphatidylethanolamine (PEG-PE), can beused as drug carriers for the antibodies of the present invention. Theemulsions are composed of two major parts: an oil core, e.g.,triglyceride, stabilized by emulsifiers, e.g., phospholipids. The pooremulsifying properties of phospholipids can be enhanced by adding abiocompatible co-emulsifier such as polysorbate 80. In a preferredembodiment, the antibody is conjugated to the surface of the lipidemulsion globules with a poly(ethylene glycol)-based, heterobifunctionalcoupling agent, poly(ethyleneglycol)-vinylsulfone-N-hydroxy-succinimidyl ester (NHS-PEG-VS).

The submicron lipid emulsion is prepared and characterized as described.Lundberg, J Pharm Sci, 83:72 (1993); Lundberg et al., Int J Pharm,134:119 (1996). The basic composition of the lipid emulsion istriolein:DPPC:polysorbate 80, 2:1:0.4 (w/w). When indicated, PEG-DPPE isadded into the lipid mixture at an amount of 2-8 mol % calculated onDPPC.

The coupling procedure starts with the reaction of the NHS ester groupof NHS-PEG-VS with the amino group of distearoylphosphatidyl-ethanolamine (DSPE). Twenty-five μmol of NHS-PEG-VS arereacted with 23 μmol of DSPE and 50 μmol triethylamine in 1 ml ofchloroform for 6 hours at 40° C. to produce a poly(ethylene glycol)derivative of phosphatidyl-ethanolamine with a vinylsulfone group at thedistal terminus of the poly(ethylene glycol) chain (DSPE-PEG-VS). Forantibody conjugation, DSPE-PEG-VS is included in the lipid emulsion at 2mol % of DPPC. The components are dispersed into vials from stocksolutions at −20° C., the solvent is evaporated to dryness under reducedpressure. Phosphate-buffered saline (PBS) is added, the mixture isheated to 50° C., vortexed for 30 seconds and sonicated with a MSE probesonicator for 1 minute. Emulsions can be stored at 4° C., and preferablyare used for conjugation within 24 hours.

Coupling of antibodies to emulsion globules is performed via a reactionbetween the vinylsulfone group at the distal PEG terminus on the surfaceof the globules and free thiol groups on the antibody. Vinylsulfone isan attractive derivative for selective coupling to thiol groups. Atapproximately neutral pH, VS will couple with a half life of 15-20minutes to proteins containing thiol groups. The reactivity of VS isslightly less than that of maleimide, but the VS group is more stable inwater and a stable linkage is produced from reaction with thiol groups.

Before conjugation, the antibody is reduced by 50 mM 2-mercaptoethanolfor 10 minutes at 4° C. in 0.2 M Tris buffer (pH 8.7). The reducedantibody is separated from excess 2-mercaptoethanol with a Sephadex G-25spin column, equilibrated in 50 mM sodium acetate buffered 0.9% saline(pH 5.3). The product is assayed for protein concentration by measuringits absorbance at 280 nm (and assuming that a 1 mg/ml antibody solutionof 1.4) or by quantitation of ¹²⁵I-labeled antibody. Thiol groups aredetermined with Aldrithiol® following the change in absorbance at 343 nmand with cystein as standard.

The coupling reaction is performed in HEPES-buffered saline (pH 7.4)overnight at ambient temperature under argon. Excess vinylsulfone groupsare quenched with 2 mM 2-mercaptoethanol for 30 minutes, excess2-mercaptoethanol and antibody are removed by gel chromatography on aSepharose CL48 column. The immunoconjugates are collected near the voidvolume of the column, sterilized by passage through a 0.45 μm sterilefilter, and stored at 4° C.

Coupling efficiency is calculated using ¹²⁵I-labeled antibody. Recoveryof emulsions is estimated from measurements of [¹⁴C]DPPC in parallelexperiments. The conjugation of reduced LL2 to the VS group ofsurface-grafted DSPE-PEG-VS is very reproducible with a typicalefficiency of near 85%.

Therapeutic Use of Multispecific Antagonists in Single and MultimodalRegimens

The present invention relates to the therapy of diverse acute andchronic inflammatory and immune-dysregulatory diseases, as well ascertain cancers, by having specific antibodies and antibodyheteroconjugates for binding to various host cells participating inimmune responses of the innate or adaptive (acquired) immunes system andby modulating the actions of critical cells and receptors by agonisticor antagonistic actions. Of particular advantage is the use of bi- andmulti-functional (bispecific or multispecific) antibodies targetingthese cells/receptors as well as target molecules of the diseasedcells/tissues. See Hansen, U.S. Pat. No. 6,458,933, incorporated hereinby reference in its entirety. Whereas Hansen focuses on clearing thehost of the pathogen (infectious organisms and cancers), the therapeuticor diagnostic agents, autoantibodies, or anti-graft antibodies, thepresent invention enables the alteration of host immunity againstcertain diseases by targeting the appropriate host cells involved inimmunity and the target cells/receptors expressed by the diseased cells.

The multispecific antagonists are formulated according to known methodsto prepare pharmaceutically useful compositions, in which thetherapeutic proteins are contained in a mixture with a pharmaceuticallyacceptable carrier. A composition is said to be a “pharmaceuticallyacceptable carrier” if its administration can be tolerated by arecipient patient. Sterile phosphate-buffered saline is one example of apharmaceutically acceptable carrier. Other suitable carriers arewell-known to those in the art. See, for example, REMINGTON'SPHARMACEUTICAL SCIENCES, 19th Ed. (1995), and later editions.

For purposes of therapy, the multispecific antagonists are administered,alone or conjugated to liposomes, to a patient in a therapeuticallyeffective amount in a pharmaceutically acceptable carrier. In thisregard, a “therapeutically effective amount” is one that isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient patient. In the present context, an agent is physiologicallysignificant if its presence results in the inactivation or killing oftargeted cells.

When therapy with naked multispecific antagonists is used, it merelyentails administering the antagonist to a subject in need of suchtreatment, and allowing sufficient time for the antagonist to bind toits targets. When therapy involves a multispecific antagonist thatincludes a hapten binding site, the subject is first administered themultispecific antagonist and then, after waiting a sufficient amount oftime for the antagonist to localize and for unbound antagonist to clearthe subject's blood stream, a carrier molecule that comprises atherapeutic agent is administered to the subject that binds to thehapten binding site on the multispecific antagonist. Alternatively,clearing agent may be administered after allowing the antagonist to bindto the target. Such pretargeting methods are described in detail, forexample, in Gautherot et al., Cancer, 80 (12 Suppl):2618-23 (1997);Karacay et al., Bioconjug Chem, 11:842-54 (2000); Sharkey et al., CancerRes, 63:354-63 (2003); Sharkey et al., Clin Can Res, 9(10 Pt2):3897S-913S; and US Patent Appln. Serial nos. 20030232011A1 and20040241158A1. If a secondary therapeutic forms part of the therapeuticregimen, it can be administered prior to, concurrently with, or afterthe multispecific antagonist is administered.

The multispecific antagonists described herein are useful for treatmentof autoimmune diseases, particularly for the treatment of Class IIIautoimmune diseases including immune-mediated thrombocytopenias, such asacute idiopathic thrombocytopenic purpura and chronic idiopathicthrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myastheniagravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever,polyglandular syndromes, bullous pemphigoid, diabetes mellitus,Henoch-Schonlein purpura, post-streptococcal nephritis, erythemanodosum, Takayasu's arteritis, Addison's disease, rheumatoid arthritis,multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis ubiterans, Sjogren'ssyndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, pamphigus vulgaris,Wegener's granulomatosis, membranous nephropathy, amyotrophic lateralsclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, perniciousanemia, rapidly progressive glomerulonephritis and fibrosing alveolitis.

The multispecific antagonists also are useful in treating inflammatoryor immune-dysregulatory disorders other than autoimmune disease.Examples of these other inflammatory or immune-dysregulatory disordersthat can be treated with composition according to the invention includesepticemia or septic shock, infection, neuropathies, graft versus hostdisease, transplant rejection, acute respiratory distress syndrome,granulomatous disease, asthma, acne, diffuse intravascular coagulation(DIC), and atherosclerosis.

In addition to their use in treating inflammatory andimmune-dysregulatory disorders, including autoimmune diseases, thetherapeutic compositions also are useful for treating a pathologicangiogenesis and cancer. Cancer includes both hematopoietic cancers,such as leukemias, lymphomas, and myelomas, and solid cancers, such ascarcinomas, melanomas, gliomas, etc. Leukemias include the myelocyticleukemias, such as AML and CML, lymphatic leukemias, such as ALL andCLL, and T-cell leukemias. Lymphomas include non-Hodgkin's lymphoma,Hodgkin's lymphoma, and T-cell lymphomas. The therapeutic compositionsalso are useful in treating the cachexia that may accompany cancer,infections, and some autoimmune diseases. In particular, multispecificantagonists according to the invention preferably include IL-6 or TNF-αas a target in this embodiment.

The multispecific antagonists also can be used in treating inflammationassociated with an infectious disease, including viral infections,bacterial infections, parasitic infections, and fungal infections.Exemplary viruses include the species of human immunodeficiency virus(HIV), herpes virus, cytomegalovirus, rabies virus, influenza virus,hepatitis B virus, Sendai virus, feline leukemia virus, Reo virus, poliovirus, human serum parvo-like virus, simian virus 40, respiratorysyncytial virus, mouse mammary tumor virus, Varicella-Zoster virus,Dengue virus, rubella virus, measles virus, adenovirus, human T-cellleukemia viruses, Epstein-Barr virus, murine leukemia virus, mumpsvirus, vesicular stomatitis virus, Sindbis virus, lymphocyticchoriomeningitis virus, wart virus and blue tongue virus. Exemplarybacteria include Anthrax bacillus, Streptococcus agalactiae, Legionellapneumophilia, Streptococcus pyogenes, Escherichia coli, Neisseriagonorrhoeae, Neisseria meningitidis, Pneumococcus, Hemophilis influenzaeB, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa,Mycobacterium leprae, Brucella abortus, Mycobacterium tuberculosis andTetanus toxin. Exemplary protozoans are Plasmodium falciparum,Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosomacruzi, Trypanosoma rhodesiensei, Trypanosoma brucei, Schistosomamansoni, Schistosoma japanicum, Babesia bovis, Elmeria tenella,Onchocerca volvulus, Leishmania tropica, Trichinella spiralis,Onchocerca volvulus, Theileria parva, Taenia hydatigena, Taenia ovis,Taenia saginata, Echinococcus granulosus or Mesocestoides corti.Exemplary mycoplasma are Mycoplasma arthritidis, Mycoplasma hyorhinis,Mycoplasma orale, Mycoplasma arginini, Acholeplasma laidlawii,Mycoplasma salivarum, and Mycoplasma pneumoniae. The fungus may be fromthe species of Microsporum, Trichophyton, Epidermophyton, Ssporothrixschenckii, Cyrptococcus neoformans, Coccidioides immitis, Histoplasmacapsulatum, Blastomyces dermatitidis, or Candida albicans. Exemplaryfungi include the species of Microsporum, Trichophyton, Epidermophyton,Ssporothrix schenckii, Cyrptococcus neoformans, Coccidioides immitis,Histoplasma capsulatum, Blastomyces dermatitidis, or Candida albicans.Exemplary parasites include malarial parasites, spirochetes and thelike, including helminthes. Listings of representative disease-causinginfectious organisms to which antibodies can be developed for use inthis invention are contained in the second and subsequent editions ofDavis et al., MICROBIOLOGY (Harper & Row, New York, 1973 and later), andare well known to one of ordinary skill in the art. In theseembodiments, the multispecific antibody preferably targets an antigenassociated with the microbe or parasite.

Sepsis and septic shock are characterized by overwhelming inflammatoryand immune responses, which make them particularly susceptible totreatment with multispecific antagonists according to the presentinvention. Treatment of these conditions according to the presentinvention entails combining agents that work via different mechanisms,and preferably by administering fusion proteins of antagonist or agonistmediators or antibodies which function against more than one targetmolecule involved in the pathogenesis of this immune dysregulatory,inflammatory disease. As advocated by Van Amersfoort et al. (ibid.), “anattempt should be made to restore the balance between the pro- andanti-inflammatory responses.” The present invention restores the balanceand provides a clear improvement art over the use of single agents thatneutralize the proinflammatory cytokines such TNF or IL-1 in patientswith sepsis, by using multispecific antagonists specific for at leasttwo different targets, where the targets are selected from the groupconsisting of proinflammatory effectors of the innate immune system,coagulation factors, and targets specifically associated with sepsis orseptic shock.

In one embodiment for treatment of sepsis or septic shock, differentanti-inflammatory agents are combined with activated protein C, as wellas with anti-coagulation agents, and at least one component of thismultiple agent therapy is an agonist or antagonist antibody to at leastone target receptor or mediator of inflammation or coagulation,including complement pathway antagonists. A listing of selectedanti-inflammatory and immunomodulating agents used to treat patientswith severe sepsis and septic shock is found in Bochud and Calandra(Brit Med J 2003; 326:262-266), and clinical trials of most of theseimmunomodulatory therapies of severe sepsis and septic shock arereviewed in Vincent et al., Clin Infect Dis 2002; 34:1084-93.

Particularly preferred agents useful in treatment of sepsis and septicshock are multispecific antagonists that target MIF, LPS, TNF-α, C5areceptor (C5aR), TLR2 or HMGB-1 as one of the targets. The other targetcan also be selected from these, as well as from other proinflammatorycytokines or receptors, such as interleukin IL-1, TSST-1 (toxic shocksyndrome toxin 1), NCA-90, NCA-95, and HLA-DR. Preferred combinations ofagents or fusion proteins for treatment of severe sepsis or septic shockinclude those that target MIF and C5a receptor (C5aR), MIF and IL-6, LPSand MIF, TNF-α and HMGB-1, TLR2 (toll-like receptor-2) and LPS, TLR2 andIL-6, TLR2 and C5aR. An anti-MIF/anti-NCA-90 or an anti-MIF/anti-HLA-DRmultispecific antagonist can be used to target granulocytes inblood/infectious deposits to neutralize MIF in patients with earlyevidence of toxic shock. Preferably these are humanized or humanantibody constructs. These are readily combined or constructed by thoseof skill in the art from available antibodies. For example, T2.5 Mab hasbeen developed as an antagonist to TLR-2 by immunizing a TLR2-neg mousewith TLR2 extracellular domain, and this antibody inhibits release ofinflammatory mediators, such as TNF-α and prevents lethal shock-likesyndrome in mice (Meng et al., J Clin Invest 2004; 113:1473-1481). Inpreferred embodiments, recombinant activated protein C is used as asecondary therapeutic in combination with antibody mixtures and fusionproteins.

It also has been discovered according to the invention that themultispecific antagonists which target both a complement regulatoryfactors such as CD46, CD55, and/or CD59 and a tumor-associated antigen,and more particularly at least bispecific antibodies in which one armtargets the complement regulatory factor and a second arm targets antumor associated antigen, are more effective in treating cancer thanantibodies that target just one of these antigens. Moreover, contrary tothe teaching of Sier et al., supra, it has been discovered that the useof beta-glucan is not obligatory in vivo for the improved efficacy of asuch a multispecific antagonist over the use of the anticancer antibodyalone, and that the bispecific antibody targeting the cancer and thecomplement-regulatory protein (e.g., CD55) increases cancer cell killingover either antibody used by itself, specifically against tumors thathave a high expression of the complement-regulatory protein (thusblocking complement-mediated cytotoxicity by antibodies).

Another preferred complement-related target for neutralizing antibodiesis complement factor H (and its variant FHL-1) involved in thealternative pathway for complement, especially since factor H may beoverexpressed by some cancers (Ajona et al., Cancer Res 2004;64:6310-6318, and references cited therein). Therefore, use ofmultispecific antagonists, and particularly multispecific antibodies,directed against complement factor H and factor FHL-1 are of particularimportance. Multispecific antagonists against complement factor H andits variant FHL-1 additionally may target CD55, CD46 and/or CD59, aswell as other complement factors. The targeting of these multispecificantagonists and to tumor-associated antigens and receptors has beenfound to enhance specific targeting of complement antibodies to thetumor cells, and to provide an advantage over use of antibodiestargeting a single antigen or epitope. This has overcome theinconsistencies in the literature published to date.

In non-malignant conditions, there is a different approach. Thisincludes neutralization or interference with other complement receptorsor factors, including complement-derived anaphylatoxin C5a orcomplement-receptor 3 (CR3, CD18/11b), which can mediate adhesion ofinflammatory cells to the vascular endothelium. In such situations,increased expression of CD46, CD55, and/or CD59 is desired in order tomitigate complement-mediated immunity, and also to reduce hyperacuterejection, as in organ transplant-rejection. Therefore, use of agonistsof such complement regulatory factors would be advantageous.

Particularly preferred agents useful in treatment of atherosclerosis aremultispecific antagonists that target MIF, low-density lipoprotein(LDL), and CEACAM6 (e.g., NCA-90). The other target can also be selectedfrom these, as well as from other proinflammatory cytokines. Preferredcombinations of agents or fusion proteins for treatment ofatherosclerosis target MIF and low-density lipoprotein-modifiedepitopes, NCA-90 and MIF, NCA-90 and low-density lipoprotein (LDL)epitopes, or LDL and CD83. There are readily combined or constructed bythose of skill in the art from commercially available antibodies. Forexample, Mab MDA2, a prototype Mab, recognizes malondialdehyde-lysingepitopes (e.g., in malondialdehyde-modified LDL) within oxidation-richatherosclerotic lesions (as described by Tsimikas et al., J Nucl Cardiol1999; 6:81-90).

In addition to sepsis and atherosclerosis, MIF has been reported to beexpressed in rabbits with atherogenesis (Lin et al., Circulation Res2000; 87:1202-1208), indicating that it is a key cytokine for thiscondition. Other diseases in which MIF has been implicated includeglomerulonephritis, arthritis, delayed-type hypersensitivity, gastricinflammation, and acute myocardial ischemia (reviewed by Yu et al., JHistochem Cytochem 2003; 625-631). Multispecific antagonists that targetMIF are therefore useful in treating any of these conditions.

As many as 500,000 individuals in the U.S. develop sepsis each year, anumber that is rising with the aging of the population. Despite the bestin antibiotic therapy and cardiopulmonary support, and the advances inunderstanding of inflammation and coagulation in sepsis, as many as halfthese cases are fatal. During infection, pro-inflammatory cytokines arereleased and activated. These include TNF-α, IL-1, and IL-6.Anti-inflammatory mediators, including IL-4 and IL-10, appearinsufficient to regulate pro-inflammatory cytokines in severe sepsis.

Prominent features of the septic response include uncontrolledinflammation and coagulation. Vascular endothelial damage is thetriggering event, whether caused by endotoxin, tissue factor, necroticcells, or amniotic fluid, becomes the triggering event. This endothelialdamage leads to release of tissue factor, which activates thecoagulation system resulting in excess thrombin generation. Subsequentclot formation promotes microvascular endothelial dysfunction, and, ifunchecked, hypoxemia, organ dysfunction, and organ failure ensue.

Endothelial damage and a shift towards a prothrombotic milieu lead todecreased expression and impaired function of endothelial receptors forthrombin, i.e., thrombomodulin, and protein C, i.e., the endothelialprotein C receptor (EPCR). Both thrombomodulin and EPCR are required forthe conversion of protein C to its active form, APC. Thus, a majorsystem for the regulation of thrombin formation, clot propagation, andprotein C activation is lost.

Nearly all patients with severe sepsis are deficient in protein C. Lowprotein C levels are associated with shock and poor outcomes, includingICU stay, ventilator dependence, and mortality. Supplying activatedprotein C exogenously in severe sepsis helps to restore regulation ofinflammatory and coagulation responses in some patients, leading to afavorable survival benefit. However, there is an obvious need for newtherapeutic modalities to reduce the procoagulant response, and preventseptic organ injury.

It has been established that blocking initiation of the procoagulantresponse before sepsis decreases mortality in nonhuman primates.Effective strategies to block initiation of extrinsic coagulation haveincluded use of monoclonal antibodies to TF, the natural TF pathwayinhibitor, and inactive analogs of FVIIa. In a recent study in baboons,it was demonstrated that blockade of the TF-VIIa complex with FVIIai atthe onset of sepsis attenuated sepsis-induced multiple organ injury anddramatically protected the lungs and kidneys. Antagonists that inhibitcomplement activation products, especially the anaphylatoxins, alsooffer promise to decrease sepsis mortality. C3a, C4a and C5a, appearduring sepsis, and the elevated anaphylotoxin plasma levels highlycorrelate with the development of multiorgan failure. In sepsis,complement may directly promote procoagulant activity or indirectlyinduce cytokine production. In vitro C5a and the terminal complex ofcomplement, C5b-9, induce tissue factor expression on endothelial cellsand monocytes, and assembly of C5b-9 on the surface of platelets hasbeen shown to stimulate prothrombinase activity. The present inventionprovides improved therapeutics for treating sepsis by providingmultispecific antagonists that target two or more of coagulationfactors, proinflammatory cytokines and complement activations products.

Additional pharmaceutical methods may be employed to control theduration of action of an antibody in a therapeutic application. Controlrelease preparations can be prepared through the use of polymers tocomplex or adsorb the antibody. For example, biocompatible polymersinclude matrices of poly(ethylene-co-vinyl acetate) and matrices of apolyanhydride copolymer of a stearic acid dimer and sebacic acid.Sherwood et al., Bio/Technology 10:1446 (1992). The rate of release ofan antibody from such a matrix depends upon the molecular weight of theprotein, the amount of antibody within the matrix, and the size ofdispersed particles. Saltzman et al., Biophys J 55:163 (1989); Sherwoodet al., supra. Other solid dosage forms are described in REMINGTON'SPHARMACEUTICAL SCIENCES, 19th ed. (1995).

The multispecific antagonists according to the invention bind to variousimmune or other host cells involved in the generation of inflammationand other immune-dysregulatory diseases (including intravascularcoagulation and myocardial ischemia). They also can be used to enhance ahost's immune response to cancer for cancer therapy or prevention. Inaddition, compositions and treatment methods are provided forneutralizing microbial toxins, such as LPS, neutralizingpro-inflammatory cytokines, and for overcoming abnormalities ofcoagulation. The methods use appropriate antibody combinations andfusion proteins directed against different participating factors in thecascade leading to severe sepsis, septic shock, and various otherimmune-dysregulatory diseases.

In general, the dosage of administered antibodies will vary dependingupon such factors as the patient's age, weight, height, sex, generalmedical condition and previous medical history. Typically, it isdesirable to provide the recipient with a dosage of antibody component,immunoconjugate or fusion protein which is in the range of from about 1pg/kg to 10 mg/kg (amount of agent/body weight of patient), although alower or higher dosage also may be administered as circumstancesdictate. These doses can be repeated as needed.

Administration of antibodies to a patient (human or domestic animal) canbe intravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, intrapleural, intrathecal, by perfusion through a regionalcatheter, or by direct intralesional injection; it may also includeinhalation, aerosols, or nasal application in certain diseases, such asasthma. When administering therapeutic proteins by injection, theadministration may be by continuous infusion or by single or multipleboluses. Intravenous injection provides a useful mode of administrationdue to the thoroughness of the circulation in rapidly distributingantibodies.

Although unconjugated multispecific antibodies and antibody fragmentsand mixtures of unconjugated antibodies and antibody fragments are thepreferred, primary therapeutic compositions for therapy according to theinvention, the efficacy of such therapy can be enhanced by supplementingthe multispecific antagonists with other therapies described herein. Insuch multimodal regimens, the supplemental therapeutic compositions canbe administered before, concurrently or after administration of themultispecific antagonists. For example, multimodal therapy of Class Mautoimmune diseases may comprise co-administration of therapeutics thatare targeted against T-cells, plasma cells or macrophages, such asantibodies directed against T-cell epitopes, more particularly againstthe CD4 and CD5 epitopes. Gamma globulins also may be co-administered.In some cases, it may be desirable to co-administer immunosuppressivedrugs such as corticosteroids and possibly also cytotoxic drugs. In thiscase, lower doses of the corticosteroids and cytotoxic drugs can be usedas compared to the doses used in conventional therapies, therebyreducing the negative side effects of these therapeutics. When thedisease to be treated is cancer, the use of various chemotherapeuticdrugs, naked antibodies used in immunotherapy, and radiation (externalor internal), can be combined with therapy according to the invention.Likewise, when infection and/or septicemia or septic shock are beingtreated, antimicrobial drugs may be used in combination with themultispecific antagonists.

In an alternative embodiment, the multispecific antagonists used fortherapy are conjugated to a drug, toxin, enzyme, oligonucleotide,hormone, hormone antagonist, immunomodulator, boron compound ortherapeutic radioisotope. Where the multispecific antagonist comprises amixture of separate antibodies, only one of the antibodies may beconjugated, or more than one of the antibodies may be conjugated. In afurther preferred embodiment, an antibody is used that comprises an armthat is specific for a low-molecular weight hapten to which atherapeutic agent is conjugated or fused. In this case, the antibodypretargets the B-cells, and the low-molecular weight hapten with theattached therapeutic agent is administered after the antibody has boundto the B-cell targets. Examples of recognizable haptens include, but arenot limited to, chelators, such as DTPA and DOTA, fluoresceinisothiocyanate, vitamin B-12 and other moieties to which specificantibodies can be raised, including also peptides and oligonucleotides.A preferred peptide is histamine-succinyl-glycine (HSG).

Therapeutically useful immunoconjugate can be obtained by conjugating aphotoactive agent or dye to an antibody fusion protein. Fluorescentcompositions, such as fluorochrome, and other chromogens, or dyes, suchas porphyrins sensitive to visible light, have been used to detect andto treat lesions by directing the suitable light to the lesion. Intherapy, this has been termed photoradiation, phototherapy, orphotodynamic therapy (Jori et al. (eds.), PHOTODYNAMIC THERAPY OF TUMORSAND OTHER DISEASES (Libreria Progetto 1985); van den Bergh, Chem Britain22:430 (1986)). Moreover, monoclonal antibodies have been coupled withphotoactivated dyes for achieving phototherapy. Mew et al., J Immunol130:1473 (1983); idem., Cancer Res 45:4380 (1985); Oseroff et al., ProcNatl Acad Sci USA 83:8744 (1986); idem., Photochem Photobiol 46:83(1987); Hasan et al., Prog Clin Biol Res 288:471 (1989); Tatsuta et al.,Lasers Surg Med 9:422 (1989); Pelegrin et al., Cancer 67:2529 (1991).Thus, the present invention contemplates the therapeutic use ofimmunoconjugates comprising photoactive agents or dyes.

Drugs which are known to act on B-cells, plasma cells and/or T-cells areparticularly useful in accordance with the present invention, whetherconjugated to the multispecific antagonist, or administered as aseparate component in combination with the multispecific antagonist.These include 5-fluorouracil, gemcitabine, methotrexate, doxorubicin,phenyl butyrate, bryostatin, cyclophosphamide, etoposide, bleomycin,doxorubicin, carmustine, vincristine, dacarbazine, procarbazine, taxol,platin derivatives, dexamethasone, leucovorin, prednisone, maytansinoidssuch as DM1, calicheamicin, rapamycin, leflunomide, FK506, immuran,fludarabine, azathiopine, mycophenolate, camptothecins (e.g., CPT-11,SN38), proteasome inhibitors (e.g., Velcade®), and cyclosporin. Drugssuch as immuran, doxorubicin, methotrexate, and fludarabine which act onboth B-cells and T-cells are particularly preferred. Illustrative oftoxins which are suitably employed in accordance with the presentinvention are ricin, abrin, ribonuclease, DNase I, Staphylococcusenterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin,Pseudomonas exotoxin, Pseudomonas endotoxin and RNAses, such asonconase. See, for example, Pastan et al., Cell 47:641 (1986), andGoldenberg, CA-A Cancer Journal for Clinicians 44:43 (1994). Othersuitable drugs and toxins are known to those of skill in the art.

Diagnostic Use of Multispecific Antagonists

Multispecific antagonists according to the invention also are useful inthe diagnosis or detection of various conditions. In the context of thisapplication, the terms “diagnosis” or “detection” can be usedinterchangeably. Whereas diagnosis usually refers to defining a tissue'sspecific histological status, detection recognizes and locates a tissue,lesion or organism containing a particular antigen. In theseembodiments, the multispecific antagonists are conjugated to adiagnostic/detection agent. The construction and administration ofdiagnostic/detection agents is described in WO 04094613 and US PublishedApplication no. 2004 0057902.

A diagnostic/detection agent is a molecule or atom, which may beadministered conjugated to the multispecific antagonist and is useful indiagnosis or detection by binding to an antigen on cells that arelocalized at the site of a disease or condition according to the presentinvention. Useful diagnostic/detection agents include, but are notlimited to, radioisotopes, dyes (such as with the biotin-streptavidincomplex), radiopaque materials (e.g., iodine, barium, gallium, andthallium compounds and the like), contrast agents, fluorescent compoundsor molecules and enhancing agents (e.g., paramagnetic ions) for magneticresonance imaging (MRI), as well as for X-rays, ultrasound and computedtomorgraphy (CT).

Preferably, the diagnostic/detection agents are selected from the groupconsisting of radioisotopes for nuclear imaging, endoscopic andintravascular detection, enhancing agents for use in magnetic resonanceimaging or in ultrasonography, radiopaque and contrast agents for X-raysand computed tomography, and fluorescent compounds for fluoroscopy,including endoscopic fluoroscopy. Fluorescent and radioactive agentsconjugated to antibodies or used in bispecific, pretargeting methods,are particularly useful for endoscopic, intraoperative or intravasculardetection of the targeted antigens associated with diseased tissues orclusters of cells, such as malignant tumors, as disclosed in GoldenbergU.S. Pat. Nos. 5,716,595 and 6,096,289, particularly with gamma-, beta-,and positron-emitters. Endoscopic applications may be used when there isspread to a structure that allows an endoscope, such as the colon,including orally-ingested cameras also used for imaging thegastrointestinal tract.

Multispecific antagonists according to the invention may comprise one ormore radioactive isotopes useful for detecting diseased tissue.Particularly useful diagnostic radionuclides include, but are notlimited to, ¹¹⁰In, ¹¹¹In, ¹⁷⁷Lu, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga,⁶⁸Ga, 86Y, ⁹⁰Y, ⁸⁹Zr, ^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I,¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ³²P, ¹¹C, ¹³N, ¹⁵O, ¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ^(52m)Mn, ⁵⁵Co,⁷²As, ⁷⁵Br, ⁷⁶Br, ^(82m)Rb, ⁸³Sr, or other gamma-, beta-, orpositron-emitters, preferably with a decay energy in the range of 20 to5,000 keV, more preferably in the range of 25 to 4,000 keV, and evenmore preferably in the range of 25 to 1,000 keV, and still morepreferably in the range of 70 to 700 keV. Radionuclides useful forpositron emission tomography include, but are not limited to: ¹⁸F, ⁵¹Mn,^(52m)Mn, ⁵²Fe, ⁵⁵Co, ⁶²Cu, ⁶⁴Cu, ⁶⁸Ga, ⁷²As, ⁷⁵Br, ⁷⁶Br, ^(82m)Rb,⁸³Sr, ⁸⁶Y, ⁸⁹Zr, ^(94m)Tc, ¹¹⁰In, ¹²⁰I, and ¹²⁴I. Total decay energiesof useful positron-emitting radionuclides are preferably <2,000 keV,more preferably under 1,000 keV, and most preferably <700 keV.Radionuclides useful as diagnostic/detection agents utilizing gamma-raydetection include, but are not limited to: ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶⁷Cu,⁶⁷Ga, ⁷⁵Se, ⁹⁷Ru, ^(99m)Tc, ¹¹¹In, ^(114m)In, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁶⁹Yb,¹⁹⁷Hg, and ²⁰¹Tl. Decay energies of useful gamma-ray emittingradionuclides are preferably 20-2000 keV, more preferably 60-600 keV,and most preferably 100-300 keV. Radioisotopes may be bound to themultispecific antagonist either directly, or indirectly by using anintermediary functional group.

The method of diagnostic imaging with radiolabeled MAbs is well-known.In the technique of immunoscintigraphy, for example, antibodies arelabeled with a gamma-emitting radioisotope and introduced into apatient. A gamma camera is used to detect the location and distributionof gamma-emitting radioisotopes. See, for example, Srivastava (ed.),RADIOLABELED MONOCLONAL ANTIBODIES FOR IMAGING AND THERAPY (Plenum Press1988), Chase, “Medical Applications of Radioisotopes,” in REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition, Gennaro et al. (eds.), pp.624-652 (Mack Publishing Co., 1990), and Brown, “Clinical Use ofMonoclonal Antibodies,” in BIOTECHNOLOGY AND PHARMACY pp. 227-49,Pezzuto et al. (eds.) (Chapman & Hall 1993). The radiation dosedelivered to the patient is maintained at as low a level as possiblethrough the choice of isotope for the best combination of minimumhalf-life, minimum retention in the body, and minimum quantity ofisotope which will permit detection and accurate measurement.

Metals are also useful in diagnostic/detection agents, including thosefor magnetic resonance imaging techniques. These metals include, but arenot limited to: gadolinium, manganese, iron, chromium, copper, cobalt,nickel, dysprosium, rhenium, europium, terbium, holmium and neodymium.In order to load an antibody component with radioactive metals,paramagnetic ions, or other detectable moieties, it may be necessary toreact it with a reagent having a long tail to which are attached amultiplicity of chelating groups for binding the ions. Such a tail canbe a polymer such as a polylysine, polysaccharide, or other derivatizedor derivatizable chain having pendant groups to which can be boundchelating groups such as, e.g., ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTPA),tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA),p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid (TETA), or NOTAor other suitable peptide, porphyrins, polyamines, crown ethers,bisthiosemicarbazones, polyoximes, and like groups known to be usefulfor this purpose. Chelates are coupled to the antibodies using standardchemistries. The chelate is normally linked to the antibody by a group,which enables formation of a bond to the molecule with minimal loss ofimmunoreactivity and minimal aggregation and/or internal cross-linking.Other, more unusual, methods and reagents for conjugating chelates toantibodies are disclosed in U.S. Pat. No. 4,824,659 to Hawthorne,entitled “Antibody Conjugates,” issued Apr. 25, 1989. Particularlyuseful metal-chelate combinations include 2-benzyl-DTPA and itsmonomethyl and cyclohexyl analogs, used with diagnostic isotopes in thegeneral energy range of 60 to 4,000 keV, such as ¹²⁵I, ¹³¹I, ¹²³I, ¹²⁴I,⁶²Cu, ⁶⁴Cu, ¹⁸F, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc, ^(94m)Tc, ¹¹C, ¹³N, ¹⁵O,⁷⁶Br, for radio-imaging. The same chelates, when complexed withnon-radioactive metals, such as manganese, iron and gadolinium areuseful for MRI, when used along with the antibodies of the invention.Macrocyclic chelates such as NOTA, DOTA, and TETA are of use with avariety of metals and radiometals, most particularly with radionuclidesof gallium, yttrium and copper, respectively. Such metal-chelatecomplexes can be made very stable by tailoring the ring size to themetal of interest. Other ring-type chelates such as macrocyclicpolyethers, which are of interest for stably binding nuclides, such as²²³Ra for RAIT are encompassed by the invention.

Contrast agents include enhancing agents for use in magnetic resonanceimaging, as well as CT contrast agents, and ultrasound contrast agents.Paramagnetic ions suitable in detection and diagnosis according to thepresent invention include chromium (III), manganese (II), iron (III),iron (II), cobalt (II), nickel (II), copper (II), neodymium (III),samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) and erbium (III.Preferred magnetic imaging agents include, for example, non-radioactivemetals, such as manganese, iron and gadolinium, complexed withmetal-chelate combinations that include 2-benzyl-DTPA and its monomethyland cyclohexyl analogs, when used along with the antibodies of theinvention, with gadolinium being particularly preferred. U.S. Pat. No.6,331,175 describes MRI technique and the preparation of antibodiesconjugated to an MRI enhancing agent. Preferred ultrasound contrastagents may comprise more than one image-enhancing agent for use inultrasound imaging. In a preferred embodiment, the contrast agent is aliposome. Preferably, the liposome comprises a bivalent DTPA-peptidecovalently attached to the outside surface of the liposome. Preferablythe liposome is gas filled.

Ions useful in other contexts, such as X-ray imaging, include but arenot limited to lanthanum (III), gold (III), lead (II), and especiallybismuth (III). Fluorescent labels include rhodamine, fluorescein andrenographin. Rhodamine and fluorescein are often linked via anisothiocyanate intermediate.

Radiopaque and contrast materials are used for enhancing X-rays andcomputed tomography, and include iodine compounds, barium compounds,gallium compounds, thallium compounds, etc. Specific compounds includebarium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid,iocetamic acid, iodamide, iodipamide, iodoxamic acid, iogulamide,iohexol, iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid,ioseric acid, iosulamide meglumine, iosemetic acid, iotasul, iotetricacid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid,ipodate, meglumine, metrizamide, metrizoate, propyliodone, and thallouschloride.

The multispecific antagonists of the present invention also can belabeled with a fluorescent compound. The presence of afluorescent-labeled MAb is determined by exposing the antibody to lightof the proper wavelength and detecting the resultant fluorescence.Fluorescent labeling compounds include fluorescein isothiocyanate,rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehydeand fluorescamine.

As in therapeutic modalities, administration of multispecificantagonists for diagnosis can be effected in a mammal by intravenous,intraarterial, intraperitoneal, intramuscular, subcutaneous,intrapleural, intrathecal, perfusion through a regional catheter, ordirect intralesional injection. When administering the antibody byinjection, the administration may be by continuous infusion or by singleor multiple boluses. Diagnosis further requires the step of detectingthe bound proteins with known techniques. A single multispecificantagonist can be used for both therapy and diagnosis/detection at thesame time.

For purposes of diagnosis and detection, the multispecific antagonistsare administered to a patient in a diagnostically effective amount in apharmaceutically acceptable carrier. In this regard, a “diagnosticallyeffective amount” is one that is capable of being detected by theequipment associated with detection once the antagonist has localizedand excess antagonist has cleared from the bloodstream.

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the presentinvention. The examples demonstrate that multispecific antagonistsaccording to the invention prevent septic shock in animal models andimprove signs and symptoms in patients with cancer-related cachexia,autoimmune disease and atherosclerotic plaques.

EXAMPLE 1 Treatment of Septic Shock

JR is a 72-year-old white male with a history of non-Hodgkin's lymphomahaving past therapy with various cytotoxic drugs, corticosteroids, aswell as Rituxan®, and presenting with stable lymphoma and a past historyof several infections that required prolonged antibiotic therapy. He isadmitted to the emergency department after being evaluated by hisgeneral practitioner with high temperature (40.7° C.), chills, dyspnea,palpitations, agitation, some confusion, and cool extremities.Examination reveals tachycardia (>90/min), hypotension (95/60 mm Hg),especially upon standing, and a reduced urine output (800 mL/d), andsigns of pneumonia. Tests show a low oxygen tension and acidosis, ablood count not detecting infection, but instead neurtopenia (3,500WBC/mL, with 10% bands), platelets of 48,000, Hg of 6 g/dL, chest x-rayreveals a generalized pneumonia, blood tests indicate reduced renalfunction, with abnormal serum creatinine (3 mg/dL) and BUN levels, andelevated serum lactate indicates tissue hypoperfusion. Blood culturesreveal the presence of S. aureus and Gram-negative bacteria, supportingthe diagnosis of septicemia. The patient is treated in the intensivecare unit for severe sepsis and septic shock, which includes generalsupportive care (oxygen), hemodynamic support by fluid infusion torestore circulating blood volume (500 mL 0.9% sodium chloride andlactated Ringer solution, with up to 2 L given over first few hours),vasopressor supportive therapy with dopamine (Intropin, 3 mcg/kg/miniv), and antibiotic therapy with 400 mg IV every 12 hrs of ciprofloxacin(Cipro). The patient is also given drotecogin alfa (activated protein C)at 24 microg/kg/hr for a total of 96 hrs. Five days after admission, thepatient is stable but does not show any significant improvement in signsor symptoms, only slightly better urine excretion, a small rise in bloodpressure, and a small drop in temperature to 39.3° C. The patient isthen given a combination of two humanized monoclonal antibodiessequentially twice weekly for 3 weeks, consisting of 300 mg anti-MIF and400 mg anti-LPS antibody, both by slow infusions over 4 hrs. During thesecond week, the patient shows less confusion, a further drop intemperature, reduction of tachycardia, dyspnea, and reduced pneumonia byboth physical exam and chest x-ray. At the end of the 3^(rd) week, hisrenal function tests also show some improvement (BUN and serumcreatinine values), and he is removed from the intensive care unit to aninfectious disease bed, with supportive care adjusted. Two months later,the patient receives a repeated cycle of activated protein C and theanti-MIF and anti-LPS antibodies, as well as a repeated course ofbroad-spectrum antibiotic, and then shows further improvement so that hebecomes ambulatory and has virtually normal mental function and anoverall 80+% reduction of pneumonia and a fever of 38.5° C., and aboutan 80% normal urine output.

EXAMPLE 2 Therapy of Systemic Lupus Erythematosus (SLE)

S.R. is a 32-year-old African-American female diagnosed 5 years earlierwith SLE, when she presented with a globerulonephritis (WHO grade 3),serositis, polyarthritis, and a vasculitic rash. She had prior therapywith corticosteroids (range of 15-60 mg per day) and hyrdoxychloroquine(200 mg/day), and at a later time also azathioprine (100 mg/day) and acourse of cyclophosphamide) because of persistent disease. Over theyears, she experienced flares of her SLE, presenting with polyarthritis,lethargy, skin rash, and serositis. She now presents with persistentlyactive disease and unresponsive to conventional therapies, but ismaintained on 40 mg prednisone daily. She is given humanized anti-CD22monoclonal antibody, epratuzumab, at 400 mg i.v. over 1 hr, repeatedonce in each of the following two weeks. Four weeks after the thirdinfusion, her circulating B-lymphocytes are reduced by 40% from baselineprior to therapy, but her Hg level has risen from 8 g/dL to 10 g/dL. Herrash and polyarthritis show some improvement, yet she requiresadditional therapy. At 8 weeks following her anti-CD22 antibody therapy,she is given a course of a bispecific antibody fusion protein consistingof a recombinant heteroconjugate of an anti-CD83 and an anti-TNF-αantibody, at a dose of 500 mg i.v. weekly×4 weeks. At evaluation at 2months later, she has a marked improvement in all organ systems, to aBILAG C and D status in most, and is capable of having her prednisonedose tapered to 7.5 mg per day. At follow-up of 3 months, most of herorgan symptoms remain stable, and she remains on this low does ofprednisone without any flare.

EXAMPLE 3 Therapy of Non-Hodgkin's Lymphoma (NHL)

SL is a 66-year-old white male with a history of diffuse large-cell NHLthat has relapsed after therapy with CHOP and rituximab, and is nowpresenting with fever, lung and mediastinal infiltrates, enlargedcervical and axillary lymph nodes, and evidence of bone marrowinvolvement based on aspiration and cytology. He receives 6 weeklyinfusions of two humanized antibodies, one against TNF-α and the otheragainst MIF, each given on the same day sequentially, over a 3-4-hrinfusion for each, at a dose of each of 450 mg. Twenty-four hours afterthe last infusion, his examination indicates that he has no majortoxicities to the therapy, and some palpable softening of his cervicaland axillary lymph nodes. At the next follow-up examination in 8 weeks,almost all of his cervical and about half of these axillary nodes havedisappeared, and his chest x-ray and CT scan show evidence of about a60% shrinkage of his pulmonary and mediastinal infiltrates. About 4months later, his examination reveals that although his lymph node andpulmonary involvement are stable, there is a suggested increase in bonemarrow involvement and a drop in his Hg to 8 g/dL. He then receives abispecific antibody consisting of a fused humanized antibody against MIFand against IL-6, given twice weekly for 3 weeks at a dose of 500 mg perslow i.v. infusion. At his 3-month evaluation, his Hg shows a rise to 11g/dL, there is a remarkable decrease of NHL cells in the bone marrowaspirate, and there are no lymph nodes palpable or disease visible inthe chest by radiological examinations. The patient's response remainsstable for another 6 months.

EXAMPLE 4 Therapy of Cancer-Related Cachexia

NR is a 58-year-old African-American male with a history of heavycigarette smoking and an inoperable non-small-cell lung cancer affectinghis left lung and paraortic and parabronchial lymph nodes on both sides.He has received combination chemotherapy, which has shownmyelotoxicities and evidence of some minor tumor shrinkage, being lessthan 40% of all measurable volume. He presents with considerable weightloss, being almost 2 meters high and now weighing 68 kg, suffering fromcancer-related cachexia. He is infused weekly for 8 weeks with ahumanized bispecific fusion antibody construct targeting both IL-6 andTNF-α, at a dose of 600 mg weekly. During the last 3 weeks, his appetiteimproves and he shows a weight gain to 75 kg at 7 weeks post therapy,with more muscle strength and generally improved vigor, which thenremains stable at 75-80 kg over the next 2 months, when he begins toshow progression of his malignant disease. Other than his cyclicchemotherapy, no corticosteroids were given during the antibody therapy,and he is considered to have responded to this treatment for cachexia.

EXAMPLE 5 Therapy of Renal Cell Carcinoma

JR is a 45-year-old white female presenting with a mass on her leftkidney and involvement of her paraortic lymph nodes on the left side anda 3×4 cm focus of disease in her upper left lung. She undergoes completeresection of her left kidney, with evacuation of adjacent lymph nodes.Six weeks later, she is given 7 weekly i.v. infusions of humanized IL-6antibody fused with IL-2 cytokine, at a dose of 500 mg (antibodyprotein), which is repeated 4 months later. At her follow-up examination3 months following the last therapy, her examination reveals a 60%reduction of her lung metastasis, and no evidence of new diseaseelsewhere.

EXAMPLE 6 Therapy of Rectal Carcinoma

PS is a 70-year-old white female with a history of total mesorectalextirpation of a rectal adenocarcinoma (T3N2) and adjuvantchemoradiation (continuous infusion 5-flurouracil and fractionated,1.8-Gy doses, five days per week over a period of 5 and a half weeks,total dose of 45 Gy external beam radiation). She does not have postsurgical chemotherapy, and 6 months later presents with 3 metastases inthe lower right lobe of the liver, ranging from 2 to 4 cm in diameter,and a serum CEA titer of 16.4 ng/mL. The primary rectal cancer isevaluated by immunohistochemistry and shows a high expression of bothCD59 and CEACAM6. She then receives 350 mg of a bispecific antibodyconsisting of a recombinantly fused humanized anti-CD59 antibody andhumanized anti-CEACAM6 antibody, given once weekly for 8 weeks,alongside a continuous infusion of fluorouracil for a period of 5 weeks.At follow-up 2 months post the last antibody infusion, the patient'sblood CEA titer drops to 12 ng/mL, but with no change by CT scans in thesize of the liver metastases. After an additional 3 months, the bloodCEA is 7 ng/mL, and there is disappearance by CT scanning of thesmallest liver metastasis and shrinkage by about 50% of the other two.The disease remains stable for another 2 months, at which time the bloodCEA level rises to 10 ng/mL, but no change yet in the size of the livermetastases.

Thus, methods and compositions for immunotherapy of inflammatory andimmune-dysregulatory diseases and cancers according to the presentinvention have been described. Many modifications and variations may bemade to the techniques and structures described and illustrated hereinwithout departing from the spirit and scope of the invention.Accordingly, it should be understood that the compositions and methodsdescribed herein are illustrative only and are not limiting upon thescope of the invention.

The contents of all documents, references and citations referencedabove, including their references, are incorporated herein in theirentirety.

What is claimed is:
 1. A multispecific antibody that reacts specificallywith at least two different targets, wherein said targets are (A) aproinflammatory effector of the innate immune system which is aproinflammatory effector cytokine or a proinflammatory effectorchemokine, wherein said antibody acts by neutralizing said (A) target,and (B) a target specifically associated with an infectious disease,acute respiratory distress syndrome, septicemia or septic shock, graftversus host disease or transplant rejection, atherosclerosis, asthma,granulomatous disease, a neuropathy, cachexia, a coagulopathy, acne,giant cell arteritis, or myocardial ischemia, wherein said latter targetis not (A), wherein CD74 is excluded as a target of said multispecificantibody, said multispecific antibody comprising a therapeutic agent. 2.A multispecific antibody that reacts specifically with at least twodifferent targets, wherein said targets are (A) a proinflammatoryeffector of the innate immune system which is a proinflammatory effectorcytokine or a proinflammatory effector chemokine, wherein said antibodyacts by neutralizing said (A) target, and (B) a target specificallyassociated with an inflammatory or immune-dysregulatory disorder,wherein said latter target is not (A), wherein CD74 is excluded as atarget of said multispecific antibody, said multispecific antibodycomprising a therapeutic agent which is anti-TNF.
 3. A multispecificantibody that reacts specifically with at least two different targets,wherein said targets are (A) a proinflammatory effector of the innateimmune system which is a proinflammatory effector cytokine or aproinflammatory effector chemokine, wherein said antibody acts byneutralizing said (A) target, and (B) a target specifically associatedwith an inflammatory or immune-dysregulatory disorder, wherein saidlatter target is not (A), wherein CD74 is excluded as a target of saidmultispecific antibody, said multispecific antibody comprising atherapeutic agent which is G-CSF.
 4. A multispecific antibody thatreacts specifically with at least two different targets, wherein saidtargets are (A) a proinflammatory effector of the innate immune systemwhich is a proinflammatory effector cytokine or a proinflammatoryeffector chemokine, wherein said antibody acts by neutralizing said (A)target, and (B) a target specifically associated with an inflammatory orimmune-dysregulatory disorder, wherein said latter target is not (A),wherein CD74 is excluded as a target of said multispecific antibody,said multispecific antibody comprising a therapeutic agent, wherein saidproinflammatory effector is a proinflammatory effector chemokine.
 5. Amultispecific antibody according to claim 4, wherein said therapeuticagent is a cytokine.
 6. A multispecific antibody that reactsspecifically with at least two different targets, wherein said targetsare (A) a proinflammatory effector of the innate immune system which isa proinflammatory effector cytokine or a proinflammatory effectorchemokine, wherein said antibody acts by neutralizing said (A) target,and (B) a target specifically associated with an inflammatory disorderor an autoimmune disease, wherein said latter target is not (A), whereinCD74 is excluded as a target of said multispecific antibody, saidmultispecific antibody comprising a therapeutic agent.
 7. Amultispecific antibody that reacts specifically with at least twodifferent targets, wherein said targets are (A) a proinflammatoryeffector of the innate immune system which is a proinflammatory effectorcytokine or a proinflammatory effector chemokine, wherein said antibodyacts by neutralizing said (A) target, and (B) a target specificallyassociated with a neuropathy, wherein said latter target is not (A),wherein CD74 is excluded as a target of said multispecific antibody,said multispecific antibody comprising a therapeutic agent.
 8. Amultispecific antibody according to claim 7, wherein said therapeuticagent is an immunomodulator.
 9. A multispecific antibody according toclaim 7, wherein target (A) is selected from the group consisting ofTNF-α, IL-1, IL-4, IL-5, IL-6, IL-8, IL-12, IL-15, IL-17 and IL-18.